Stabilized insulinotropic peptides and methods of use

ABSTRACT

The present invention provides stably cross-linked insulionotropic polypeptides having superior and un-expected benefits in the treatment of conditions involving abnormal glucose homeostasis, e.g., type 2 diabetes and conditions relating to type 2 diabetes. Such benefits include, but are not limited to, extended polypeptide half-life, enhanced alpha-helicity, improved thermal stability and protease resistance, increased functional activity and pharmacologic properties, improved bioavailability when administered by any route, and improved bioavailability and gastrointestinal absorption when delivered orally, as compared to the corresponding unmodified polypeptides. The invention also provides compositions for administering the polypeptides of the invention, as well as methods for pre-paring and evaluating the polypeptides of the invention.

REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. Provisional Application Ser.No. 61/363,097, filed Jul. 9, 2010. This application is also related toPCT International Application No. PCT/US2009/000438, filed Jan. 23, 2009and entitled “Compositions and Methods for the Treatment of ViralInfections,” and PCT International Application No. PCT/US2010/039223,filed Jun. 18, 2010 and entitled “Structured Viral Peptide Compositionsand Method of Use,” which in turn claims priority from U.S. ProvisionalPatent Application Ser. No. 61/218,209, filed on Jun. 18, 2009. Each ofthe aforementioned applications are herein incorporated by reference intheir entireties.

Any and all references cited in the text of this patent application,including any U.S. or foreign patents or published patent applications,International patent applications, as well as, any non-patent literaturereferences, including any manufacturer's instructions, are herebyexpressly incorporated herein by reference.

BACKGROUND

Diabetes refers to a disease process resulting in abnormal glucosehomeostasis that is derived from multiple causative factors andcharacterized by elevated levels of glucose in the blood (i.e.,hyperglycemia). Persistent or uncontrolled hyperglycemia is associatedwith increased and premature morbidity and mortality. Often abnormalglucose homeostasis is also associated both directly and indirectly withalterations of the lipid, lipoprotein and apolipoprotein metabolism andother metabolic and hemodynamic diseases. Therefore patients with type 2diabetes mellitus are at especially increased risk of macrovascular andmicrovascular complications, including coronary heart disease, stroke,peripheral vascular disease, hypertension, nephropathy, neuropathy, andretinopathy. Therefore, therapeutical control of glucose homeostasis,lipid metabolism and hypertension are critically important in theclinical management and treatment of diabetes mellitus.

There are two generally recognized forms of diabetes. In type 1diabetes, or insulin-dependent diabetes mellitus (IDDM), patientsproduce little or no insulin, the hormone which regulates glucoseutilization. In type 2 diabetes, or noninsulin dependent diabetesmellitus (NIDDM), patients often have plasma insulin levels that are thesame or even elevated compared to nondiabetic subjects; however, thesepatients have developed a resistance to the insulin stimulating effecton glucose and lipid metabolism in the main insulin-sensitive tissues,which are muscle, liver and adipose tissues, and the plasma insulinlevels, while elevated, are insufficient to overcome the pronouncedinsulin resistance.

Insulin resistance is not primarily due to a diminished number ofinsulin receptors but to a post-insulin receptor binding defect that isnot yet understood. This resistance to insulin responsiveness results ininsufficient insulin activation of glucose uptake, oxidation and storagein muscle and inadequate insulin repression of lipolysis in adiposetissue and of glucose production and secretion in the liver.

The available treatments for type 2 diabetes have recognizedlimitations. While physical exercise and reductions in dietary intake ofcalories will dramatically improve the diabetic condition, compliancewith this treatment is very poor because of well-entrenched sedentarylifestyles and excess food consumption, especially of foods containinghigh amounts of saturated fat. Problems with compliance of various knownantidiabetic agents also exist due to a lack of overwhelming patientacceptance of injection as the main mode of delivery.

With type 2 diabetes, increasing the plasma level of insulin byadministration of sulfonylureas (e.g. tolbutamide and glipizide) ormeglitinide, which stimulate the pancreatic beta-cells to secrete moreinsulin, and/or by injection of insulin when sulfonylureas ormeglitinide become ineffective, can result in insulin concentrationshigh enough to stimulate the very insulin-resistant tissues. However,dangerously low levels of plasma glucose can result from administrationof insulin or insulin secretagogues (sulfonylureas or meglitinide), andan increased level of insulin resistance due to the even higher plasmainsulin levels can occur. The biguanides increase insulin sensitivityresulting in some correction of hyperglycemia. However, the twobiguanides, phenformin and metformin, can induce lactic acidosis andnausea/diarrhea. Metfourmin has fewer side effects than phenformin andis often prescribed for the treatment of type 2 diabetes.

Despite these known therapies, there is no generally applicable andconsistently effective means of maintaining an essentially normalfluctuation in glucose levels in type 2 diabetes. Additional methods oftreating the disease, including alternative therapeutic interventions(e.g., incretin-based therapies, such as GLP-1-receptor agonists andDPP-4 inhibitors) and improved modes of pharmacologic administration(e.g., sublingual, intranasal, intratracheal, inhalation, and oraladministration) to improve drug utility and compliance, are still underinvestigation.

The incretin system—a recognized possible point of intervention fordiabetic therapies—includes glucagon-like peptide (GLP-1) andglucose-dependent insulinotropic polypeptide (GIP) (“incretins”), whichtogether play an important role in the regulation of insulin secretionby the pancreas and glucose production by the liver. In addition, theincretins are recognized as playing an important role in maintainingpancreatic β-cell mass and differentiation, preventing β-cell apoptosis,decreased glucagon secretion, deceleration of gastric emptying, andpromotion of early satiety leading to weight loss.

Normal glucose levels in the blood are, in part, regulated by a balanceof the actions of insulin (causes a reduction in blood glucose) andglucagon (signals liver to produce glucose). The balanced action betweenthese two hormones is maintained in a normal individual throughpancreatic β-cell production of insulin and glucagon in response toplasma glucose levels. The incretins provide an additional layer ofregulation on glucose levels which is triggered at the time of foodingestion. GLP-1 and GIP are released from cells of the intestine uponfood intake, which stimulate insulin secretion via GLP-1-receptors andGIP receptors on precreatic cells, and in the case of GLP-1, also whichinhibits glucagon secretion from the pancreas, thereby decreasingglucose production by the liver and lowering blood glucose levelsoverall.

In type 2 diabetes, however, incretin system is greatly diminished.Specifically, the insulinotropic and glucagon-reducing effects of GLP-1and GIP are impaired in individuals with type 2 diabetes. Thisrecognition led to the recent development of incretin-based therapies,including GLP-1-receptor agonists, such as liraglutide and others, andincretin mimetics, such as exenatide, which interact with the GLP-1receptor and other receptors of the incretin system to promote insulinrelease and block glucagon secretion, thereby lowering the overallplasma glucose level. See Gutniak, M., et al. N. Engl. J. Bled. 1992;326:1316-1322; Grossman, S., “Differentiating incretin therapies basedon structure, activity, and metabolism: Focus on Liraglutide,”Pharmacotherapy, 2009; 29(12):25S-32S.

One incretin-based therapy under development includes the GLP-1-receptoragonist and GLP-1 analog, glucagon-like insulinotropic peptide (GLIP),which is a fragment of GLP-1. Gutniak et al., 1992. In normal subjects,the infusion of GLIP significantly lowered the meal-related increases inblood glucose concentration, and the plasma concentrations of insulinand glucagon. In patients with NIDDM, the treatment reduced therequirement for insulin by 8 fold. In patients with IDDM, the GLIPtreatment lowered the insulin required by one half. Thisglucose-dependent activity is a very desirable characteristic for atherapeutic agent that can be used to treat type 2 diabetes whileavoiding complications of hypoglycemic side effects.

A more recently developed incretin-based therapy for treating type 2diabetes is exenatide, which was approved by the Federal Food and DrugAdministration (FDA) as a subcutaneous injection (under the skin) of theabdomen, thigh, or arm, 30 to 60 minutes before the first and last mealof the day. Exenatide—a synthetic version of exendin-4, a hormone foundin the saliva of the Gila monster that was first isolated by Dr. JohnEng in 1992 (Eng, J. et al., J. Biol. Chem. 267:742-7405 (1992)) anddescribed in U.S. Pat. No. 5,424,286 to Eng displays human glucagon-likepeptide-1 (GLP-1) activities, functioning as a regulator of glucosemetabolism and as an insulinotropic agent (i.e., increases insulinrelease) through its agonistic action at the GLP-1-receptor.

According to the FDA package insert, exenatide enhancesglucose-dependent insulin secretion by the pancreatic β-cells,suppresses inappropriately elevated glucagon secretion, and slowsgastric emptying, although the mechanism of action is still under study.Exenatide is a 39-amino-acid peptide and an insulin secretagogue withglucoregulatory effects which binds and activates the pancreatic GLP-1receptor (GLP-1R) with similar affinity and potency as GLP-1 and therebypromotes insulin secretion and blocks glucagon secretion in aglucose-dependent manner. The effects of exenatide also reportedlyinclude slowing of gastric emptying to modulate nutrient absorption,reduction of food intake and body weight and increased pancreatic β-cellmass and function. In addition, it is inherently a poor substrate fordegradation by dipeptidyl peptidase-IV (DPP-IV)—the normal degradativeenzyme responsible for removal of the incretins (GLP-1 and GIP).Exenatide was approved by the FDA on Apr. 28, 2005 for patients whosediabetes was not well-controlled on other oral antibiabetic agents (e.g.metformin, sulfonylureas, thiazolidinediones).

Exenatide raises insulin levels quickly (within about ten minutes ofadministration) with the insulin levels subsiding substantially over thenext hour or two. A dose taken after meals has a much smaller effect onblood sugar than one taken beforehand. The effects on blood sugardiminish after 6-8 hours. The medicine is available in two doses: 5 mcgand 10 mcg. Treatment often begins with the 5 mcg dosage, which isincreased if adverse effects are not significant.

Two important limitations on the use of exenatide and otherincretin-based polypeptide antidiabetic therapeutics potentially include(1) relatively short half-lives upon administration (e.g., exenatide's2.5 hour half life when delivered by the approved intravenous route) dueto proteolytic degradation and (2) lack of effective, but less invasive(and thereby more patient compliant), alternative administration routes(e.g., orally, sublingually, or intranasally) that provide forsufficient bioavailability (Gedulin et al., “Pharmacokinetics andpharmacodynamics of exenatide following alternative routes ofadministration,” Int'l J Pharmaceuticals, 356 (2008) 231-238).

Accordingly, the development of optimized incretin-based or otherinsulinotropic polypeptide therapeutics, such as, optimized exenatide,that are imparted with superior stability, protease resistance, andpharmacologic properties, as well as the development of suchtherapeutics that can be delivered successfully (i.e., achievingimproved bioavailability, gastrointestinal absorption and pharmacologicproperties) by alternative, more patient compliant delivery routes(e.g., orally deliverable form of exenatide) would be significantadvances in the art.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the surprising discoverythat stably crosslinking a polypeptide having an insulinotropicactivity, including incretin hormones (e.g., glucagon-like peptide-1),incretin analogs (e.g., liraglutide) or incretin mimetics (e.g.,exenatide), provide superior and unexpected benefits in the treatment ofconditions involving abnormal glucose homeostasis, e.g., type 2 diabetesand conditions relating to type 2 diabetes. Such benefits include, butare not limited to, extended half-life, enhanced alpha-helicity,improved thermal stability and protease resistance, increased functionalactivity and pharmacologic properties, improved bioavailability whenadministered by any route, and improved bioavailability andgastrointestinal absorption when delivered orally, as compared touncrosslinked counterparts. Thus, the invention provides a new andadvantageous approach to the administration of insulinotropicpolypeptides that provides benefits not previously obtained oravailable. Accordingly, the present invention enables improved half-life(and thus, bioavailability, effectiveness, etc.) of insulinotropicpolypeptides delivered by injection-based routes. Additionally, theinstant invention makes it feasible to deliver effective amounts (i.e.,which are bioavailable upon delivery) of insulinotropic polypeptideagents (e.g., GIP, GLP-1, GLP-2, exenatide, liraglutide, taspoglutide,or albiglutide) via non-injection-based routes, including oral,intranasal, and sublingual routes, thereby improving the chances ofbetter patient compliance and ease of use.

Accordingly, in one aspect, the invention providesstructurally-fortified insulinotropic polypeptide agents, includingincretin hormones (e.g., glucagon-like peptide-1), analogs (e.g.,liraglutide) or mimetics (e.g., exenatide) for use in treating orpreventing type 2 diabetes and conditions associated with type 2diabetes, wherein said agents possess extended half-life, enhancedalpha-helicity, improved thermal stability and protease resistance,increased functional activity and pharmacologic properties, and improvedbioavailability when administered by any route.

In another aspect, the present invention provides methods andcompositions for structurally-fortifying insulinotropic polypeptideagents, including incretin hormones, analogs and mimetics (e.g.,exenatide) to impart an extended half-life, enhanced alpha-helicity,improved thermal stability and protease resistance, increased functionalactivity and pharmacologic properties, and improved bioavailability whenadministered by any route.

In yet another aspect, the present invention provides therapeuticmethods for treating or preventing type 2 diabetes or conditionsassociated with type 2 diabetes by administering a therapeuticallyeffective amount of a insulinotropic polypeptide agent of the inventionvia any route (e.g., as injectable or oral agent). The insulinotropicpolypeptide agents of the invention can include or be based onfull-length incretin hormones, analogs or mimetics, or any suitablefunctional fragment or variant thereof which retains biologicalactivity.

In yet another embodiment, the invention provides structurallyconstrained polypeptides having amino acids 1-39 of the exenatide. Incertain embodiments, the amino acids 1 to 39 of the exenatide peptidecan be contiguous amino acids in the primary sequence of the peptide. Incertain embodiments, the amino acids are adjacent to each other e.g.,the amino acids are present on the same face of the helix, in at leastone native state of the peptide sequence in the context of the fulllength polypeptide. For example, the adjacent amino acids can be presentin a single stacked column of amino acids in a helix, or in adjacentstacks of amino acids in a single face of the structured helix. In anembodiment, the structurally constrained polypeptides include at leastone modification from the group consisting of: hydrocarbon staple, aminoacid mutation, and non-natural amino acid incorporation. In certainembodiments, the structurally constrained polypeptides include 2, 3, 4,5 or more modifications. In certain embodiments, the constrainedpolypeptides comprise various hydrocarbon staples including, but notlimited to, pairing selected from the group consisting of an S5-S5pairing (ie. i, i+4), an S5-R8 pairing (i.e. i, i+7), an S8-R5 pairing(i.e. i, i+7), an R3-S6 pairing (ie. i, i+3), an R6-S3 pairing (ie. i,i+3), an R3-S5 pairing (ie. i, i+3), an R5-S3 pairing (ie. i, i+3), orcombinations of pairings within the polypeptide sequence.

In another embodiment, amino acids 1 to 39 of exenatide comprises atleast 3 contiguous amino acids, or at least two amino acids on a singleface of a helix, or at least two interacting face amino acids; or aconservative substitution thereof. A single face of a helix comprisesone, two, three, or four adjacent stacked columns of amino acids whereinthe stacked columns of amino acids are defined by positions a, d, and g;positions b and e; or positions c and f; in an alpha helix having 7amino acids per two turns wherein the amino acids are consecutively andserially assigned positions a-g (see, e.g., FIG. 3); and positions a andd; positions b and e; or positions c and f in a 3¹⁰ helix having 2 aminoacids per two turns wherein the amino acids are consecutively andserially assigned positions a-f; or homologues thereof.

The invention in other embodiments provides exenatide and otherbioactive insulinotropic polypeptides for use in the invention. Thestructurally constrained exenatide peptides of the invention can includeamino acids 1-39 of SEQ ID NO: 1 comprising a hydrocarbon staple betweenpositions 24 and 28 (i.e., SAH-Ex(B) of FIG. 7B) and optionally ahydrocarbon staple between positions 10 and 14 (i.e., SAH-Ex(A) of FIG.7B) and at least 3 contiguous amino acids, or at least two amino acidson a single face of a helix, or at least two interacting face aminoacids of GLP-1 (see FIG. 2A) or homologues thereof. The structurallyconstrained peptides of the invention can include the exenatide or GLP-1sequence only, or the exenatide or GLP-1 sequence flanked on theC-terminus, or the N-terminus, or both. The peptides provided by theinvention can further include non-amino acid modifications in additionto modifications to structurally constrain the peptides. For example,peptides can include functional groups for targeting of the peptides invivo, or to alter the pharmacokinetic and/or pharmacodynamic propertiesof the peptide. Such modifications are known in the art.

Amino acid positions that constitute a stacked column of amino acids canbe defined by positions corresponding to positions on a peptide sequencehelical wheel of SEQ ID NO: 1 (see FIG. 3). The sequence of thestructurally constrained peptide can be aligned with the sequences ofother homologous insulinotropic peptides, including GIPP, GIP, GLP-1precursor, GLP-1, GLP-2, liraglutide, taspoglutide, and albiglutideMethods for performing sequence alignments are well known to those ofskill in the art. Further, corresponding amino acids in helices can bedetermined using any of a number of publicly available coil detectionprograms. In reference to the sequence provided in SEQ ID NO: 1, thestacked columns of amino acids can include those depicted in FIG. 3.

The invention also provides polypeptides having at least 3 interactingface amino acids or a conservative substitution of an interacting faceamino acid, from the exenatide polypeptide sequence of SEQ ID NO: 1, orhomologues thereof. The interacting face of the polypeptide is a singleface of the peptide wherein the interacting face amino acids areselected from positions corresponding to amino acids from SEQ ID NO:1such as H1, D9, F22, I23, and L26, or other critical residues known tobe important for biological activity such as G4, F6, T7, G30, P31, P36,P37, P38, as defined by structural determination (PDB ID 3C5T) and/oralanine scanning of SEQ ID NO: 1. The structurally constrained peptidesprovided by the invention can include additional amino acid sequences,either other sequences from exenatide or other insulinotropicpolypeptides. The additional amino acid sequences may or may not bestructurally constrained. The invention provides for the use ofexenatide or homologous sequences having at least 3 contiguous aminoacids of an exenatide or homologous peptide, or at least two amino acidson a single face of a helix of an exenatide or homologous peptide, or atleast two interacting face amino acids of an exenatide or homologouspeptide; or a conservative substitution thereof. A single face of ahelix of the exenatide or homologous peptide includes one, two, three,or four adjacent stacked columns of amino acids wherein the stackedcolumn of amino acids is defined by positions a, d, and g; positions band e; or positions c and f; in an alpha helix, wherein position a is anamino acid in the helix, and the amino acids are consecutively andserially assigned letters a through g in an alpha helix; or homologuesthereof.

For example, an alpha-helix and a stacked column of amino acids of apeptide is defined as positions 1, 4, 5, 8, 11, 12, 15, 18, 19, 22, 25,26, 29, 32, and 33; or positions 2, 5, 6, 9, 12, 13, 16, 19, 20, 23, 26,27, 30, 33, and 34; or positions 3, 6, 7, 10, 13, 14, 17, 20, 21, 24,27, 28, 31, and 34; or positions 4, 7, 8, 11, 14, 15, 18, 21, 22, 25,28, 29, 32, and 35; or positions 5, 8, 9, 12, 15, 16, 19, 22, 23, 26,29, 30, and 33; or positions 6, 9, 10, 13, 16, 17, 20, 23, 24, 27, 30,31, and 34; or 7, 10, 11, 14, 17, 18, 21, 24, 25, 28, 31, 32, and 35 ofSEQ ID NO: 1 or homologues thereof. As provided herein, a single face ofa peptide having an alpha-helical structure can include one, two, three,or four adjacent stacked columns of amino acids.

The invention further provides peptides having the interacting faceamino acids of the exenatide or homologous peptide. The interacting faceis an example of one face on the helical peptides provided by theinstant invention. Amino acids of an interacting face include aminoacids corresponding to positions such as H1, G2, T5, S8, D9, K12, E15,E16, V19, R20, F22, I23, L26, K27 of SEQ ID NO:1 or may be furtherlimited to amino acids corresponding to positions H1, D9, F22, I23, andL26 on SEQ ID NO: 1.

In further embodiments, the invention provides any of the structurallyconstrained peptides of the invention in a pharmaceutically acceptablecarrier. The invention further provides a peptide of the invention in apharmaceutical carrier in a unit dosage form. The invention providesstructurally constrained peptides of the invention functionally linkedto a carrier. In certain embodiments, the carrier includes a protein orlipid to alter the pharmacokinetics and/or pharmacokinetic properties ofthe structurally constrained peptide. In certain embodiments, thestructurally constrained peptide of the composition is functionallylinked to a carrier protein in a specified orientation as established bya site-directed linkage.

In still further embodiments, the invention provides methods for theprevention, amelioration, or treatment of type 1 or type 2 diabetes, forexample in a subject, by administration of a structurally constrainedpeptide of the invention to the subject in a therapeutically effectiveamount. The method can further include one or more of identifying asubject as being in need of prevention, amelioration, or treatment ofdiabetes, or monitoring the subject for the prevention, amelioration, ortreatment of diabetes. In certain embodiments, the invention providesmethods of prevention, amelioration, and treatment of diabetes whereinthe method includes at least one of promoting euglycemia by enhancinginsulin release.

The invention provides for the preparation of a medicament including astructurally constrained peptide of the invention delivered byinjection, inhalation, or orally to promote euglycemia. The medicamentscan be for the prevention and/or treatment of diabetes.

In certain embodiments, the invention provides kits including at leastone of a structurally constrained peptide of the invention andinstructions for use.

In one aspect, the invention provides a structurally-fortifiedinsulinotropic polypeptide comprising an alpha helix and one or moremolecular tethers, wherein each molecular tether covalently couples asingle pair of residues residing on the alpha helix of said polypeptide,thereby structurally fortifying the insulinotropic polypeptide.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a structurally-fortified insulinotropicpolypeptide of the invention and one or more pharmaceutically acceptableexcipients. In still a further aspect, the invention provides a methodfor treating or preventing diabetes comprising administering atherapeutically effective amount of a structurally-fortifiedinsulinotropic polypeptide of the invention or a composition comprisinga structurally-fortified insulinotropic polypeptide of the invention.

In an embodiment, the structurally-fortified insulinotropic polypeptideof the invention can be exenatide, GIPP, GIP, GLP-1 precursor, GLP-1,GLP-2, GLP-1 (7-37), GLP-1-(7-36), liraglutide, taspoglutide,albiglutide or LY2189265.

In another embodiment, the structurally-fortified insulinotropicpolypeptide of the invention can be exenatide.

In yet another embodiment, the number of molecular tethers perpolypeptide can be between 1-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9 or2-10.

In still another embodiment, the at least one residue pair resideswithin the N-terminal half of the polypeptide.

The at least one residue pair can reside within the C-terminal half ofthe polypeptide.

The at least one residue pair can reside at any position along thepolypeptide.

In yet another embodiment, the polypeptide comprises at least onecoupled residue pair within the C-terminal half of the polypeptide andanother coupled residue within the N-terminal half of the polypeptide.

In still another embodiment, the polypeptide corresponds to an exenatidehaving any one of SEQ ID NOs: 2 or 15-38.

In still a further embodiment, the polypeptide corresponds to an GLP-1having any one of SEQ ID NOs: 39-62.

In another embodiment, the polypeptide has a first molecular tetherlocated at position (i, i+3), or (i, i+4) or (i, i+7) relative to theresidue positions of the alpha helix of the polypeptide. The polypeptidemay have a second molecular tether located at position (i, i+3), or (i,i+4) or (i, i+7) relative to the residue positions of the alpha helix ofthe polypeptide, with the proviso that the first and second moleculartethers are not located at identical positions.

In certain embodiments, where at least two molecular tethers areemployed, the at least two molecular tethers may be configured in a“stitched” arrangement. Molecular tethers which are “stitched” refers towhere sequentially-arranged linkages are made from a common origin. Thatis, the use of “stitched” cross-links is where double linkages are madefrom a common origin (e.g., X1, X5, and X9, where X5 is the anchor pointfor both staples). Thus, the invention encompasses the incorporation ofone or more crosslinks within a polypeptide sequence to either furtherstabilize the sequence or facilitate the structural stabilization,proteolytic resistance, thermal stability, acid stability, pharmacologicproperties, and biological activity enhancement of longer polypeptidestretches. FIG. 5B shows an embodiment of polypeptides having multiplecross-links or molecular tethers which are in a “stitched”configuration.

Accordingly, in an embodiment, the tethered polypeptides of theinvention may comprise a pair of molecular tethers in a stitchedconfiguration whereby the end of the first molecular tether and thebeginning of the second molecular tether originate at a common residuein the polypeptide.

In still another embodiment, the fortified polypeptide possesses ahalf-life that is at least 2-fold, or 3-fold, or even 8-fold greaterthan the half-life of a non-fortified counterpart polypeptide.

In yet another embodiment, the fortified polypeptide possesses aresistance to chymotrypsin in vitro that is at least 2-fold greater, or3-fold greater, or event 8-fold greater than the resistance tochymotrypsin in vitro of a non-fortified counterpart polypeptide.

In still another embodiment, the fortified polypeptide possesses aresistance to chymotrypsin in vitro that is at least 6-fold greater, or13-fold greater than the resistance to chymotrypsin in vitro of anon-fortified counterpart polypeptide.

In a still further embodiment, the fortified polypeptide possesses aresistance to a serum protease in vivo that is at least 2-fold, or5-fold or 10-fold greater than the resistance to the serum protease invivo of a non-fortified counterpart polypeptide.

In certain embodiments, the method of treating diabetes of the inventioninvolves administering the polypeptides of the invention via an oraldelivery route.

In certain other embodiments, the method of treating diabetes of theinvention involves administering the polypeptides of the invention viaan injection-based delivery route.

Other embodiments of the invention will be understood base on thedisclosure provided infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings, in which:

FIG. 1 (A) provides the polypeptide sequences of the insulinotropicpolypeptide agents, exendin-4, exenatide, Gastric Inhibitory PolypeptidePreprotein (GIPP), Gastric Inhibitory Peptide (GIP), Glucagon-likePeptide-1 Precursor (GLP-1P), Glucagon-like Peptide-1 (GLP-1),Glucagon-like Peptide-2 (GLP-2), fragments of GLP-1, liraglutide,taspoglutide, albiglutide and LY2189265, which are exemplaryinsulinotropic polypeptides that can be used as templates in variousembodiments of the invention for structural fortification by hydrocarbonstapling. (B) provides a sequence alignment of the human glucagonpeptide subfamily, highlighting critical amino acid residues thatinteract with GLP-1 receptor and conserved residues. The residues ofexendin-4(9-39) that interact with GLP-1R are colored blue (i.e., 15E,19V, 20K, 22F, 23I, 26L, 27K, 32S). Conserved residues are coloredyellow (i.e., residue positions 4, 6, 21, 24, 25). Positions where onlyfour residues are conserved are colored green (i.e., positions 1, 5, 8,9, 11). (Runge S et al. 2008 J Biol Chem 283:11340-11347).

FIG. 2 (A) shows the structure of the GLP-1 receptor in complex with thealpha-helical region of exenatide (PDB ID 3C5T), and highlights thoseamino acids, including residues of the interacting face shown in balland stick mode, which were determined by alanine scan to be especiallyimportant for functional activity (Runge, S. et al., 2008, J. Biol.Chem., 283: 11340-11347). An example of substitution sites fornon-natural amino acid insertion on the non-interacting face of the coreexenatide alpha-helix are also shown in space-fill mode to demonstratethe design of a doubly stapled exenatide peptide. The figure furtherprovides the amino acid sequences of exendin-4 and residues 9-33 ofexendin-4 shown with highlights of the above-mentioned critical aminoacids as determined by alanine scan of the interacting face of exenatide(indicated in orange, underline and marked with a “*”) and the aminoacid residues marking the attachment points of the hydrocarbon staples(indicated in magenta, underlined and marked with a “A”). (B) providesthe amino acid sequence of GLP-1 (7-37) and indicates critical aminoacid residues for functional activity (underlined). (Contillo et al.Proceedings of the 16^(th) American Peptide Symposium Jun. 26-Jul. 1,1999, Minneapolis, Minn., U.S.A.).

FIG. 3 shows a helical wheel depiction of the alpha-helical region ofexenatide, highlighting the location of the face of the exenatide helixthat interacts with the GLP-1 receptor. Substitution sites for stapleinsertion can be localized to the non-interacting face of the helix toenhance alpha-helical structure, receptor binding affinity, andfunctional activity. The particular residue positions of thenon-interacting face side that may be involved in forming an attachmentpoint of a hydrocarbon staple is not limited, and can include residueslocated at the N-terminus, C-terminus, or at any location between theends of the polypeptide.

FIG. 4 shows how non-natural amino acids bearing hydrocarbon tethers(e.g., olefin tethers) can be inserted into the peptide sequence togenerate singly stapled peptides. Staples can be located at (A) (i,i+3), (B) (i, i+4), and (C) (i, i+7) positions along the length of thepeptide helix to generate a library of singly stapled peptides (i.e.“staple scan”) to identify optimal positioning for the desiredbiophysical and biological properties. (D) Further illustrates staplescanning to identify the optimal staple position(s) for achieving thedesired biophysical, biological, and pharmacologic properties of apolypeptide agent of interest (e.g., exenatide). Staple scanninginvolves the sequential evaluation of staple positions along the lengthof the peptide sequence template. An i, i+4 staple scan starting at theN-terminus is shown.

FIG. 5A shows how multiple staples of similar or different compositionscan be inserted along the length of the peptide alpha-helix to generateddoubly, triply, or multiply stapled peptides. Staples can be located at(i, i+3), (i, i+4), and (i, i+7) positions along the length of thepeptide helix to generate a library of multiply stapled peptides (i.e.“staple scan”) to identify optimal positioning for the desiredbiophysical and biological properties. (A-F) Examples of multiplystapled peptides using various combinations of i, i+3, i, i+4, and i,i+7 crosslinks. (G, H) The optimal staple positions for achieving thedesired biophysical, biological, and pharmacologic properties can beidentified by staple scanning, which involves the sequential evaluationof discrete staple positions in combination along the length of thepeptide sequence template. (G) A double i, i+4 staple scan starting atthe N- and C-termini; (H) A triple i, i+4 staple scan that includesvariable positioning of a third staple located at the middle of thepeptide sequence and between the N- and C-terminal staples.

FIG. 5B shows further examples of structurally-stabilized insulinotropicpeptides using sequentially installed staples such that the N- andC-terminal staples conjoin at a central non-natural amino acid bearingtwo olefinic side changes in the α, α position. Configurations includesequential i, i+3 (I), i+4 (J), and i, i+7 staples (K), and combinationsthereof, such as tandem i, i+3/i, i+4 (L), i, i+3/i, i+7 (M), i, i+4/i,i+3 (N), i, i+4/i, i+7 (O), i, i+7/i, i+3 (P), and i, i+7/i, i+4 (O)staples. As described above in FIG. 5A, the optimal staple positioningto achieve the desired biophysical, biological, and pharmacologicproperties can be identified by staple scanning, which involves thesequential placement of discrete tandem staples (I-Q) along the lengthof the peptide sequence template, as represented by the arrows.

FIG. 6 shows a synthetic chemistry schema (A) for the syntheses ofolefinic non-natural amino acids employed in the generation of i, i+3staples. (B) Depicts examples of olefinic non-natural amino acids.

FIG. 7 (A) Compositions of Stabilized Alpha-Helices (“SAH”) of Exenatide(SAH-Ex) and GLP-1 (SAH-GLP1). A mutant construct of exenatide(Met14NorLeu) was used as the template for the construction of singlyand doubly stapled SAH-Ex peptides. (B) Exemplary singly and doublystapled SAH-Ex peptides employed in biophysical, biological, andpharmacologic studies. X, crosslinking non-natural amino acid; B,norleucine.

FIG. 8 provides a circular dichroism spectra of the template (A) andSAH-Ex (B-D) polypeptides across a broad temperature range,demonstrating the enhanced alpha-helicity of singly and doubly stapledSAH-Ex peptides. (E) Provides temperature melt curves for the templateand SAH-Ex peptides demonstrating the enhanced thermal stability ofsingly and doubly stapled SAH-Ex peptides. Specifically, (A)-(D) showsthe circular dichroism spectra of Ex(Met14NorLeu), SAH-Ex(A), SAH-Ex(B),and SAH-Ex(A,B), highlighting the increased alpha-helicity of the singlyand doubly stapled peptides across a broad temperature range (5° C.-83°C.). (E) shows a circular dichroism temperature melt plot forEx(Met14NorLeu), SAH-Ex(A), SAH-Ex(B), and SAH-Ex(A,B), demonstratingthe enhanced thermal stability of the singly and doubly stapled peptidescompared to the corresponding unstapled template peptide.

FIG. 9 demonstrates the enhancement of protease resistance by single-and double-stapling of the template exenatide peptide (SAH-Ex).Specifically, singly and doubly stapled SAH-Ex peptides are moreresistant to chymotrypsin (pH 7) compared to the unmodified exenatidetemplate peptide. In this example, the singly stapled peptides exhibit2-3 fold enhancement in chymotrypsin resistance compared to the templatepeptide, whereas the doubly stapled peptide displays 8-fold enhancementcompared to the template peptide and 2.7-4 fold enhancement compared tothe corresponding singly stapled peptides.

FIG. 10 demonstrates that singly and doubly stapled SAH-Ex peptides aremore resistant to pepsin (pH 2) compared to the unmodified exenatidetemplate peptide. In this example, SAH-Ex(B) exhibits 6 fold enhancementin pepsin resistance compared to the template peptide, whereas thedoubly stapled peptide displays 13 fold enhancement compared to thetemplate peptide and 2-13 fold enhancement compared to the correspondingsingly stapled peptides.

FIG. 11 illustrates the results of an ELISA-based assay for thedetection of stapled SAH-Ex of the invention. Specifically, the graphdemonstrates that an exenatide ELISA-based assay kit detects seriallydiluted unmodified Ex(Met14NorLeu) and doubly-stapled SAH-Ex(A,B)polypeptides. (Exendin-4 (Heloderma suspectum), EIA Kit PhoenixPharmaceuticals, Inc. EK-070-94). As demonstrated, exenatide ELISA assayrecognizes the template mutant exenatide peptide and its doubly stapledderivative, enabling the measurement of SAH-Ex levels in plasma.

FIG. 12 demonstrates detection of full-length doubly stapled SAH-Expeptide in the plasma of mice treated by either oral gavage orintravenous injection. Equivalent levels of full-length SAH-Ex peptidewere detected for both routes of administration. Mice treated withvehicle only served as a negative control.

FIG. 13 demonstrates the mechanism of proteolytic resistance conferredby insertion of hydrocarbon staples. (A) Insertion of the two pairs ofolefinic non-natural amino acids without crosslinking (e.g. UnstapledAlpha Helix of gp41: UAH-gp41₍₆₂₆₋₆₆₂₎(A,B)) does not confer significantprotection from chymotrypsin proteolysis. However, upon olefinmetathesis, the corresponding doubly stapled analog,SAH-gp41₍₆₂₆₋₆₆₂₎(A,B), exhibited an 8-fold longer half-life thanUAH-gp41₍₆₂₆₋₆₆₂₎(A,B), indicating that the staples themselves arerequired to confer the striking protease resistance. (B) UAH- andSAH-gp41₍₆₂₆₋₆₆₂₎(A,B) displayed similar circular dichroism meltingprofiles, with T_(m) values of 27° C. and 22° C., respectively.Temperature-dependent unfolding was reversible for both peptides, asevidenced by the overlapping repeat melting curves. These datademonstrate that overall alpha-helical stabilization, which is similarfor the two constructs, does not account for the striking proteaseresistance of SAH-gp41₍₆₂₆₋₆₆₂₎(A,B). In addition, the reversibility ofunfolding highlights the absence of peptide aggregation, which likewisecannot account for the striking protease resistance ofSAH-gp41₍₆₂₆₋₆₆₂₎(A,B). (C) Comparative chymotrypsin degradationpatterns of unmodified, singly stapled, doubly stapled, and 4-placesubstituted but unstapled peptides. These data demonstrate that theN-terminal staple uniquely prevented proteolytic hydrolysis of thecleavage site flanked by the staple, with no corresponding M+18 speciesobserved by LC/MS analysis. The C-terminal staple slowed, rather thancompletely blocked, proteolysis at sites upstream of the staple. The4-place substituted but unstapled derivative UAH-gp41₍₆₂₆₋₆₆₂₎(A,B) wasnot capable of blocking proteolysis at the position flanked by theN-terminal pair of non-natural amino acids, nor slow the rate ofproteolysis as effectively as the C-terminal singly stapled peptide(T_(1/2) 77 min for SAH-gp41₍₆₂₆₋₆₆₂₎(B); T_(1/2) 36 min forUAH-gp41₍₆₂₆₋₆₆₂₎(A,B)). The doubly stapled peptideSAH-gp41₍₆₂₆₋₆₆₂₎(A,B) synergistically benefited from theanti-proteolysis features of both the N-terminal and C-terminal staples.(D) Comparative ¹H NMR analysis of SAH-gp41₍₆₂₆₋₆₆₂₎(A,B) and thecorresponding unmodified template peptide, T649v. The indole protons(˜10.6 p.p.m) corresponding to the two N-terminal tryptophan residues ofT649v are represented by two sharp peaks in T649v, consistent with fastexchange between multiple conformations. In contrast, the indole protonpeaks in the ¹H NMR spectrum of SAH-gp41₍₆₂₆₋₆₆₂₎(A,B) are broadened andsplit, reflective of a discretely structured N-terminus as a result ofpeptide stapling. Although this mechanistic dissection specificallyinvolved doubly-stapled HIV-related polypeptides, similaranti-proteolysis and structural reinforcement results were obtained forthe doubly stapled insulinotropic polypeptides of the invention (FIGS.8-10).

FIG. 14 demonstrates that structurally-stabilized and protease resistantSAH-Ex (A) and SAH-GLP1 (B) peptides stimulate glucose-stimulatedinsulin release from isolated pancreatic islets. These data highlightthat hydrocarbon stapling of SAH-Ex and SAH-GLP 1 peptides preservesfunctional activity while maximizing structural stability and proteaseresistance, yielding pharmacologically optimized insulin secretagogues.

DETAILED DESCRIPTION

The instant invention relates to the unexpected finding that stablycrosslinking a polypeptide having an insulinotropic activity, includingincretin hormones (e.g., glucagon-like peptide-1), incretin analogs(e.g., liraglutide) or incretin mimetics (e.g., exenatide), providessuperior and unexpected benefits in the treatment of conditionsinvolving abnormal glucose homeostasis, e.g., type 2 diabetes andconditions relating to type 2 diabetes. Such benefits include, but arenot limited to, extended half-life, enhanced alpha-helicity, improvedthermal stability and protease resistance, increased functional activityand pharmacologic properties, improved bioavailability when administeredby any route, and improved bioavailability and gastrointestinalabsorption when delivered orally, as compared to uncrosslinkedcounterparts. Thus, the invention provides a new and advantageousapproach to the administration of insulinotropic polypeptides thatprovides benefits not previously obtained or available. Accordingly, thepresent invention enables improved half-life (and thus, bioavailability,effectiveness, etc.) of insulinotropic polypeptides delivered byinjection-based routes. Additionally, the instant invention makes itfeasible to deliver effective amounts (i.e., which are bioavailable upondelivery) of insulinotropic polypeptide agents (e.g., GIP, GLP-1, GLP-2,exenatide, liraglutide, taspoglutide, or albiglutide) vianon-injection-based routes, including oral, intranasal, and sublingualroutes, thereby improving the chances of better patient compliance andease of use.

Accordingly, the present invention is directed to compositions, kits andmethods utilizing and/or making the structurally constrainedinsulinotropic polypeptides, including structurally constrained incretinhormones (e.g., glucagon-like peptide-1), incretin analogs (e.g.,liraglutide) or incretin mimetics (e.g., exenatide) for use in treatingand/or preventing conditions involving abnormal glucose homeostasis,e.g., type 2 diabetes and conditions relating to type 2 diabetes.

The invention is based, at least in part, on the results provided hereindemonstrating that hydrocarbon-stapled, alpha-helical exenatide peptidesand functional variants thereof display alpha-helical structuralreinforcement, neutral and acid protease resistance, thermal stability,and enhanced pharmacologic properties, including oral absorption,remedying the classical shortcomings of lengthy peptide therapeutics. Asa result, the peptides have superior pharmacologic properties in vivocompared to their unmodified counterparts, reducing the frequency andquantity of a structurally constrained peptide that needs to beadministered as compared to an unmodified polypeptide sequence. Invarious embodiments, the polypeptides of the invention comprise one ormore alpha helix domain(s) that are stabilized with at least onemolecular tether, e.g., hydrocarbon staple, but may include two, threeor more such hydrocarbon staples. The inclusion of multiple hydrocarbonstaples is particularly suited for alpha helical peptides that are 20 ormore amino acids in length. The inclusion of more than one (e.g., 2, 3,4, 5, depending on the length of the peptide) hydrocarbon staplesprovides for exceptional structural, neutral and acid proteaseresistance, and thermal stability of the modified polypeptides, yieldingbioactive peptides with strikingly enhanced pharmacologic properties invivo.

As demonstrated herein, the stapled insulinotropic polypeptides of theinvention (e.g., stapled exenatide) demonstrate stabilizedalpha-helicity, proteolytic and thermal stability, and more desirablepharmacokinetic and bioavailability properties than a comparablenon-structurally constrained polypeptides. Importantly, as furtherdemonstrated herein, the stapled polypeptides of the invention canwithstand protease action during oral delivery and gastrointestinaltract exposure, which is not possible for certain FDA-approvedintravenous insulinotropic agents (e.g., exenatide or liraglutide).

DEFINITIONS

It is understood that this invention is not limited to the particularmaterials and methods described herein. It is also to be understood thatthe terminology used herein is for the purpose of describing particularaspects and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, protocols, reagents and vectors which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that such citedreferences are prior art.

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise. For example, areference to “a host cell” includes a plurality of such host cells knownto those skilled in the art.

An “agent” is understood herein to include a therapeutically activecompound or a potentially therapeutic active compound. An agent can be apreviously known or unknown compound. As used herein, an agent istypically a non-cell based compound, however, an agent can include abiological therapeutic agent, e.g., polypeptide (e.g., insulinotropicpolypeptide) or nucleic acid therapeutic, cytokine, antibody, etc. Anagent can include the single or multiply stapled insulinotropicpolypeptide (e.g., exenatide), and any functional homologs, fragments,analogs, derivatives or mimetics thereof.

As used herein, the term “functional fragment” refers to a polypeptidefragment of any insulinotropic polypeptide of the invention, includingincretin hormones (e.g., glucagon-like peptide-1), incretin analogs(e.g., liraglutide) or incretin mimetics (e.g., exenatide), whichretains at least about 10%, or greater than about 20% or 30%, or 40%, or50%, or 60%, or 70%, or 80%, or 90%, or at least about 99% or more ofthe biologic or pharmacologic activity or properties of itscorresponding native insulinotropic polypeptide, e.g., exenatide orGLP-1. The fragment can be of any contiguous region of amino acids ofthe polypeptide on which it is based. The fragment may also be in theform of a chimeric sequence of two or more different contiguous regionsof amino acids of the polypeptide on which it is based which are joinedtogether to form the fragment. In the context of the present invention,an example of a fragment of a naturally-occurring insulinotropicpolypeptide (e.g., GLP-1) is residues 1-20 of GLP-1 (which can bedenoted GLP-1₁₋₂₀ or by any similarly used nomenclature that would beapparent to the skilled artisan.

As used herein, the term “analog” or “analogue” refers to a compound X(e.g., a polypeptide, peptide or amino acid) which retains chemicalstructures of X necessary for functional activity of X yet which alsocontains certain chemical structures which differ from X. In the contextof the present invention, an example of an analogue of anaturally-occurring insulinotropic polypeptide is a GLP-1 polypeptidewhich includes one or more non-naturally-occurring amino acids, e.g.,those amino acid changes involved in the hydrocarbon staples of theinvention. The analogs of the invention preferably retain at least about10%, or greater than about 20% or 30%, or 40%, or 50%, or 60%, or 70%,or 80%, or 90%, or at least about 99% or more of the biologic orpharmacologic activity or properties of its corresponding nativeinsulinotropic polypeptide, e.g., exenatide or GLP-1.

As used herein, a “mimetic” of a compound X refers to a compound inwhich chemical structures of X necessary for functional activity of Xhave been replaced with other chemical structures which mimic theconformation of X. Examples of peptidomimetics include peptidiccompounds in which the peptide backbone is substituted with one or morebenzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science260:1937-1942), peptides in which all L-amino acids are substituted withthe corresponding D-amino acids and “retro-inverso” peptides (see U.S.Pat. No. 4,522,752 by Sisto). Mimetics, in the context of the invention,may also include natural or synthetic polypeptides that bear little orno sequence homology and can be of very different overlength to acounterpart polypeptide against which it is compared, whereby themimetic has at least about 10%, or greater than about 20% or 30%, or40%, or 50%, or 60%, or 70%, or 80%, or 90%, or at least about 99% ormore of the biologic or pharmacologic activity or properties of acounterpart polypeptide against which it is compared. For example,exenatide is a mimetic of GLP-1, having little overall sequence (53%identity) and an extended C-terminal region, but shares biologicalactivity of GLP-1 at the GLP-1-receptor.

As used herein, a “derivative” of a compound X (e.g., a peptide or aminoacid) refers to a form of X in which one or more reaction groups on thecompound have been derivatized with a substituent group. Examples ofpeptide derivatives include peptides in which an amino acid side chain,the peptide backbone, or the amino- or carboxy-terminus has beenderivatized (e.g., peptidic compounds with methylated amide linkages).

As used herein, the term “functional exenatide variant,” “exanatide-likepeptide,” “exanatide homologue,” or “functional variant thereof” (ofexenatide) is meant to refer to any peptide having a similar orhomologous sequence to exenatide and having at least 50%, or 60%, or70%, or 80%, or 90%, or 95%, or even 99% or more of the activity of theexenatide peptide of SEQ ID NO: 1, as measured by any suitable means forassaying exenatide activity.

As used herein, the term “insulinotropic polypeptide” or “insulinotropicagent” refers to a polypeptide or small molecule or other compound whichhave an insulinotropic activity. As used herein, a polypeptide having an“insulinotropic activity” is defined as one which stimulates or affects,either directly or indirectly, the production (e.g., expression,release, secretion, formation, etc.) and/or activity and/or amount ofinsulin. For example, an insulinotropic polypeptide can refer to onewhich causes, either directly or indirectly, a release of insulin from3-cells of the pancreas and/or causes, either directly or indirectly, anincrease in the level of insulin in the plasma. Insulinotropicpolypeptides can include any naturally occurring or syntheticpolypeptide, and can further including any functional fragment,derivative, variant, homologue, or mimetic thereof. Insulinotropicpolypeptides can include components of the incretin system (e.g., GLP-1,GIP) and any analogs, functional fragments, derivatives or mimeticsthereof. The insulinotropic activity can be glucose-dependent orglucose-independent. The insulinotropic polypeptides of the inventionmay have additional or other activities as well and the term is notmeant to be limiting in any manner as to other such activities. Forexample, an insulinotropic polypeptide of the invention, e.g., exenatideor GLP-1, can also block or minimize, either directly or indirectly, theproduction of glucagon. The term, “insulinotropic activity,” is notmeant to be tied to any specific mechanism of action. For example,DPP-IV is a regulatory degredative enzyme of the incretin system whichfunctions as the normal degradative enzyme of GLP-1 and GIP. A mutantDPP-IV could be insulinotropic if its degradative activity is block orreduced such that the effective amount of GLP-1 and GIP—both whichstimulate insulin production—is increased. Thus, such a mutant DPP-IVwould be insulinotropic because it indirectly results in an increase inthe amount of insulin.

It will be also appreciated that the insulinotropic agents orpolypeptides (i.e., having an insulinotropic activity) can be regardedas agents or polypeptides having a “glucoregulatory” or “glycemiccontrol” activity. Thus, the present invention also contemplatespolypeptides having a glucoregulatory or glycemic control activity. Asused herein, the term “glucoregulatory” or “glycemic control activity”refers to an activity that directly or indirectly is involved in, isresponsive to or controls the metabolism of glucose in the body. Thus,the present invention is not limited to polypeptides having aninsulinotropic activity, but instead may extend to any polypeptide thatis glucoregulatory or has a glycemic control activity, which mayinclude, for example, any polypeptide that affects, either directly orindirectly, the production (e.g., expression, release, secretion,formation, etc.) and/or activity and/or amount of glucose, insulin, orglucagon and the like.

As used herein “amelioration” or “treatment” is understood as meaning tolessen or decrease at least one sign, symptom, indication, or effect ofa specific disease or condition, e.g., type 2 diabetes or relatedcondition thereof. For example, amelioration or treatment of type-2diabetes can be to stabilize blood glucose levels, Amelioration andtreatment can require the administration of more than one dose of anagent, either alone or in conjunction with other therapeutic agents andinterventions. Amelioration or treatment do not require that the diseaseor condition be cured.

As used herein, the term “amino acid” refers to a molecule containingboth an amino group and a carboxyl group. Suitable amino acids include,without limitation, both the D- and L-isomers of the 20 common naturallyoccurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G,H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letterabbreviations)) as well as the naturally occurring and non-naturallyoccurring amino acids including beta-amino acids, prepared by organicsynthesis or other metabolic routes and that can be applied forspecialized uses such as increasing chemical diversity, functionality,binding capacity, structural mimesis, and stability.

As used herein, the term “amino acid side chain” or “amino acid R group”refers to a moiety attached to the α-carbon in an amino acid. Forexample, the amino acid side chain or R group for alanine is methyl, theamino acid side chain for phenylalanine is phenylmethyl, the amino acidside chain for cysteine is thiomethyl, the amino acid side chain foraspartate is carboxymethyl, the amino acid side chain for tyrosine is4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acidside chains are also included, for example, those that occur in nature(e.g., an amino acid metabolite) or those that are made synthetically(e.g., an alpha di-substituted amino acid, a beta-amino acid).

As used herein, “carrier protein” for use with a structurallyconstrained peptide of the instant invention in a composition isunderstood as a protein or other substance such as a lipid that whenconjugated to the constrained peptide elicit an enhanced insulinproduction. Examples of carrier conjugates are known in the art andcould include for example a molecule which targets the singly ormultiply stapled exenatide peptide or its homologues to the appropriatereceptors.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator to be detected at a level that is statistically different thana sample from a normal, untreated, or control sample. Control samplesinclude, for example, cells in culture, one or more laboratory testanimals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art. An analytecan be a naturally occurring substance that is characteristicallyexpressed or produced by the cell or organism (e.g., an antibody) or asubstance produced by a reporter construct (e.g, β-galactosidase orluciferase). Depending on the method used for detection the amount andmeasurement of the change can vary. Changed as compared to a controlreference sample can also include decreased binding of a ligand to areceptor, e.g., to a pancreatic GLP-1 receptor, in the presence of anantibody, antagonist, or other inhibitor. Determination of statisticalsignificance is within the ability of those skilled in the art.

As used herein, the term “co-administration” as used herein isunderstood as administration of one or more agents to a subject suchthat the agents are present and active in the subject at the same time.Co-administration does not require a preparation of an admixture of theagents or simultaneous administration of the agents.

As used herein, the term “conservative amino acid substitution” is onein which the amino acid residue is replaced with an amino acid residuehaving a similar side chain. For example, families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Other conserved amino acidsubstitutions can also occur across amino acid side chain families, suchas when substituting an asparagine for aspartic acid in order to modifythe charge of a peptide. Thus, a predicted nonessential amino acidresidue in the exenatide polypeptide (SEQ ID NO: 1), for example, can bereplaced with another amino acid residue from the same side chain familyor homologues across families (e.g. asparagine for aspartic acid,glutamine for glutamic acid). Conservative changes can further includesubstitution of chemically homologous non-natural amino acids (i.e. asynthetic non-natural hydrophobic amino acid in place of leucine, asynthetic non-natural aromatic amino acid in place of tryptophan).

As used herein, the term “contacting a cell” is understood herein asproviding an agent to a test cell e.g., a cell to be treated in cultureor in an animal, such that the agent or isolated cell can interact withthe test cell or cell to be treated, potentially be taken up by the testcell or cell to be treated, and have an effect on the test cell or cellto be treated. The agent or isolated cell can be delivered to the celldirectly (e.g., by addition of the agent to culture medium or byinjection into the cell or tissue of interest), or by delivery to theorganism by an enteral or parenteral route of administration fordelivery to the cell by circulation, lymphatic, or other means.

As used herein, “detecting”, “detection” and the like are understoodthat an assay performed for identification of a specific analyte in asample, a product from a reporter construct in a sample, or an activityof an agent in a sample (e.g., binding inhibition, inhibition ofsyncytia formation, infectivity inhibition). Detection can include thedetermination of the level of a stapled peptide in the blood by ELISAkit or LC/MS analysis, the functional effect of the stapled peptide on aphysiologic read-out such as serum glucose level, serum insulin level,or hemoglobin A1C percentage. The amount of analyte or activity detectedin the sample can be within the range of detection for the particularassay, none, or below the level of detection of the assay or method.

By “diagnosing” as used herein refers to a clinical or other assessmentof the condition of a subject based on observation, testing, orcircumstances for identifying a subject having a disease, disorder, orcondition based on the presence of at least one sign or symptom of thedisease, disorder, or condition. Typically, diagnosing using the methodof the invention includes the observation of the subject for other signsor symptoms of the disease, disorder, or condition, e.g. type 2diabetes.

The terms “effective amount,” or “effective dose” or a “therapeuticallyeffective amount or dose” refers to that amount of an agent to producethe intended pharmacological, therapeutic or preventive result. Thepharmacologically effective amount results in the amelioration of one ormore signs or symptoms of type 2 diabetes or related disorders. Forexample, a therapeutically effective amount preferably refers to theamount of a therapeutic agent that decreases blood glucose levels, by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or more as compared to anuntreated control subject. The evaluation of whether an amount is aneffective amount can be done using any standardized testing known tothose of ordinary skill in the art in the area of diabetes, includingmeasuring changes in blood glucose levels, increasing levels of seruminsulin, decreasing hemoglobin A1C percentage (i.e., the long termmeasure of glycemic status and diabetes control). More than one dose ofan agent may be required to provide an effective dose.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. On theother hand, the term “ineffective” indicates that a treatment does notprovide sufficient pharmacological effect to be therapeutically useful,even in the absence of deleterious effects, at least in the unstratifiedpopulation. (Such a treatment may be ineffective in a subgroup that canbe identified by the expression profile or profiles). “Less effective”means that the treatment results in a therapeutically significant lowerlevel of pharmacological effectiveness and/or a therapeutically greaterlevel of adverse physiological effects, e.g., greater liver toxicity.

Thus, in connection with the administration of a drug, a drug which is“effective against” a disease or condition indicates that administrationin a clinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such as aimprovement of symptoms, a cure, a reduction in disease signs orsymptoms, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating the particular type of disease or condition.

As use herein, the “face” of a helix, for example, an alpha-helix or a3₁₀ helix, is understood as the amino acids that are “stacked” in ahelix of a protein so that when the helix is positioned vertically, theamino acids in a single face are depicted as being one on top of theother (see, e.g., FIG. 3). For example, an alpha-helix has about 3.6amino acids per turn. Therefore, when a peptide having a sequenceabcdefga′b′c′d′e′f′g′ forms an alpha helix, the fourth and fifth aminoacids (i+3 and i+4), i.e., amino acids d and e, will “stack” over thefirst amino acid (position 1+˜3. 6 amino acids), and the eighth aminoacid, amino acid a′ (i+7), will stack over amino acid a to form a faceof the helix and starting a new turn with amino acid a′ (see, e.g., FIG.3). In an alpha-helix, amino acid b, the second amino acid, will “stack”with the fifth and sixth amino acids, i.e., amino acids e and fat the +3and +4 positions, and with amino acid b′ at the +7 position to form aface of the helix. Faces on helices starting with amino acid c, d, e, f,and g can be readily determined based on the above disclosure.Furthermore, a face of a helix can include two adjacent, three adjacent,or four adjacent columns of “stacked” residues.

An example of a “face” of a helix includes the “interacting face” of thehelix (e.g., FIG. 3 shows the interacting face as that face of exenatideknown to bind to the GLP-1-receptor). An “interacting face” amino acidresidue is a residue that makes contact with the receptor, see e.g.,FIGS. 2 and 3, when altered from the wild-type sequence of thepolypeptide, results in abolishing or substantially abolishing thepolypeptide functional activity. Substantially abolishing is understoodas reducing the functional activity of an exenatide or functionallyhomologous peptide to less than about 50%, less than about 40%, lessthan about 30% of the wild-type peptide in an appropriate assay (e.g.,receptor binding assay, functional assays that monitor second messengersignaling or changes in phosphorylation status of effectors downstreamof receptor signaling, or assays that monitor glucose levels orglucose-stimulated insulin release in vitro or in vivo). The interactingface amino acid residues of the exenatide and exenatide-like peptidescan readily be determined by methods well known in the art, such asstructural determination and alanine scanning, as reported for the GLP-1receptor and a subfragment of exenatide (amino acids 9-33) as picturedin FIG. 2. The term “interacting face” amino acid residue as usedherein, includes conservative substitutions of the interacting faceamino acids that do not disrupt function of the sequence. Generally, the“interacting face” amino acid residues are found at the interacting faceof the alpha helix (as defined in FIGS. 2 and 3 for the core exenatidealpha-helix).

It will be apparent to those of ordinary skill in the art that someinsulinotropic polypeptides (e.g., exenatide) or their functionalhomolog residues are less prone to substitution while others are moreaccepting of changes, as can be determined, for example, by “alanine orstaple scanning,” as described further herein. The insulinotropicpolypeptides such as exenatide, GLP-1, and liraglutide, containhomologous sequences and alpha-helical domains that are readilyidentifiable by those possessing ordinary skill in the art by sequencebased homology, structural homology and/or functional homology. Suchmethods are well known in the art and include bioinformatics programsbased on pairwise residue correlations (e.g.,ch.embnet.org/software/COILS_form.html), which have the ability torecognize coiled coils from protein sequences and model their structures(See Lupas, A., et al. Science 1991. 252(5009); p. 1162-1164).

As used herein, the term “hydrocarbon stapling”, refers to a process forstably cross-linking a polypeptide having at least two modified aminoacids that helps to conformationally bestow the native secondarystructure of that polypeptide. Hydrocarbon stapling promotes ormaintains a helical secondary structure in a peptide predisposed to havean helical secondary structure, e.g., alpha-helical secondary structure,to attain or maintain its native alpha-helical conformation. Thissecondary structure increases resistance of the polypeptide toproteolytic cleavage and heat, and also may increase hydrophobicity.Alternative nomenclature may be used to refer to “hydrocarbon stapling,”including “hydrocarbon tethering or crosslinking,” “moleculartethering,” “intraresidue stapling, tethering, or crosslinking”“intrapeptidyl stapling, tethering, or crosslinking” and the like.

Hydrocarbon stapling promotes and maintains an alpha-helical secondarystructure in peptides that thermodynamically favor an alpha-helicalstructure. As noted, this fortification of the polypeptide's secondarystructure increases resistance of the polypeptide to proteolyticcleavage (e.g., by chymotrypsin or other proteases of thegastrointestinal tract) and heat, and also may increase thehydrophobicity of the polypeptide. Accordingly, the hydrocarbon stapled(and consequently, structurally constrained and fortified) polypeptidesof the invention as described herein have improved biological activityrelative to a corresponding non-hydrocarbon stapled (not structurallyconstrained) polypeptide, including extended half-life, enhancedalpha-helicity, improved thermal stability and protease resistance,increased functional activity and pharmacologic properties, and improvedbioavailability when administered by any route.

The hydrocarbon stapled polypeptides can include one or more tethers(linkages) between two non-natural amino acids, which tether(s)significantly enhances the helical secondary structure of thepolypeptide. Generally, to promote a helical structure, the tetherextends across the length of one or two helical turns (i.e., about 3-3.6or about 7 amino acids). Accordingly, amino acids positioned at i andi+3; i and i+4; or i and i+7 are ideal candidates for chemicalmodification and cross-linking. Thus, for example, where a peptide hasthe sequence . . . X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , and theamino acid X is independently selected for each position, cross-linksbetween X1 and X4, or between X1 and X5, or between X1 and X8 are usefulas are cross-links between X2 and X5, or between X2 and X6, or betweenX2 and X9, etc. The use of multiple cross-links (e.g., 2, 3, 4 or more)is also contemplated. The use of multiple cross-links is effective atstabilizing and optimizing the peptide, especially with increasingpeptide length, as is the case for some lengthy peptides such asinsulinotropic exenatide and exantide-like peptides. Thus, the inventionencompasses the incorporation of more than one crosslink within thepolypeptide sequence. The use of multiple crosslinks is effective atstabilizing and optimizing the peptide, especially with increasingpeptide length. The invention also encompasses the incorporation of oneor more crosslinks within a polypeptide sequence, where the crosslinksare formed in a “stitched” configuration, i.e., wherein twosequentially-occurring staples (or crosslinks) arise from a commonorigin residue (e.g., as depicted in FIG. 5B).

As used herein, the term “staple scan” or “staple scanning” refersinvolves the sequential evaluation of staple positions along the lengthof the peptide sequence template. In one embodiment, staple scanning maybe achieved by synthesis of a library of stapled peptides whereby thelocation of the i and i+3; i and i+4; and i and i+7 single and multiplestaple stitches (which may be in a stitched configuration) arepositioned sequentially down the length of the peptide sequence, andsampling all possible positions to identify desired or optimalproperties and activities for the stapled or stitched constructs.

Methods for forming intramolecular tethers, e.g., hydrocarbon staple,can be found, for example, in U.S. Publication No. 2006/0014675A1,200610008848A1, 2004/0171809A1 and U.S. Pat. Nos. 7,723,469, 7,192,713,and 7,084,244, each of which are incorporated herein by reference.Intramolecular tethers can include one or more of an ether, thioether,ester, amine, or amide moiety. In some cases, a naturally occurringamino acid side chain can be incorporated into the tether. For example,a tether can be coupled with a functional group such as the hydroxyl inserine, the thiol in cysteine, the primary amine in lysine, the acid inaspartate or glutamate, or the amide in asparagine or glutamine.Accordingly, it is possible to create a tether using naturally occurringamino acids rather than using a tether that is made by coupling twonon-naturally occurring amino acids. It is also possible to use a singlenon-naturally occurring amino acid together with a naturally occurringamino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary alpha-helical structure, whereas, in some instances, it isdesirable to provide less constraint on the secondary alpha-helicalstructure, and thus a longer tether may be desired.

Additionally, while examples of tethers spanning from amino acids i toi+3, i to i+4; and i to i+7 have been described in order to provide atether that is primarily on a single face of the alpha helix, thetethers can be synthesized to span any combinations of numbers of aminoacids.

In some instances, alpha disubstituted amino acids are used in thepolypeptide to improve the stability of the alpha helical secondarystructure. However, alpha disubstituted amino acids are not required,and instances using mono-alpha substituents (e.g., in the tethered aminoacids) are also envisioned.

As used herein, the terms “identity” or “percent identity”, refers tothe subunit sequence similarity between two polymeric molecules, e.g.,two polynucleotides or two polypeptides. When a subunit position in bothof the two molecules is occupied by the same monomeric subunit, e.g., ifa position in each of two peptide's is occupied by serine, then they areidentical at that position. The identity between two sequences is adirect function of the number of matching or identical positions, e.g.,if half (e.g., 5 positions in a polymer 10 subunits in length), of thepositions in two peptide or compound sequences are identical, then thetwo sequences are 50% identical; if 90% of the positions, e.g., 9 of 10are matched, the two sequences share 90% sequence identity. The identitybetween two sequences is a direct function of the number of matching oridentical positions. Thus, if a portion of the reference sequence isdeleted in a particular peptide, that deleted section is not counted forpurposes of calculating sequence identity. Identity is often measuredusing sequence analysis software e.g., BLASTN or BLASTP (available at(www.ncbi.nih.gov/BLAST). The default parameters for comparing twosequences (e.g., “Blast”-ing two sequences against each other), byBLASTN (for nucleotide sequences) are reward for match=1, penalty formismatch=−2, open gap=5, extension gap=2. When using BLASTP for proteinsequences, the default parameters are reward for match=0, penalty formismatch=0, open gap=11, and extension gap=1. Additional, computerprograms for determining identity are known in the art.

As used herein, “isolated” or “purified” when used in reference to apolypeptide means that a naturally polypeptide or protein has beenremoved from its normal physiological environment (e.g., proteinisolated from plasma or tissue) or is synthesized in a non-naturalenvironment (e.g., artificially synthesized in an in vitro translationsystem or using chemical synthesis). Thus, an “isolated” or “purified”polypeptide can be in a cell-free solution or placed in a differentcellular environment (e.g., expressed in a heterologous cell type). Theterm “purified” does not imply that the polypeptide is the onlypolypeptide present, but that it is essentially free (about 90-95%, upto 99-100% pure) of cellular or organismal material naturally associatedwith it, and thus is distinguished from naturally occurring polypeptide.Similarly, an isolated nucleic acid is removed from its normalphysiological environment. “Isolated” when used in reference to a cellmeans the cell is in culture (i.e., not in an animal), either cellculture or organ culture, of a primary cell or cell line. Cells can beisolated from a normal animal, a transgenic animal, an animal havingspontaneously occurring genetic changes, and/or an animal having agenetic and/or induced disease or condition.

As used herein, “kits” are understood to contain at least onenon-standard laboratory reagent for use in the methods of the invention.For example, a kit can include at least one of, preferably at least twoof at least one peptide for modification, one or more aldehyde moleculesfor modification of peptides, and instructions for use, all inappropriate packaging. The kit can further include any other componentsrequired to practice the method of the invention, as dry powders,concentrated solutions, or ready to use solutions. In some embodiments,the kit comprises one or more containers that contain reagents for usein the methods of the invention; such containers can be boxes, ampules,bottles, vials, tubes, bags, pouches, blister-packs, or other suitablecontainer forms known in the art. Such containers can be made ofplastic, glass, laminated paper, metal foil, or other materials suitablefor holding reagents.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially altering its functional activity and/or secondarystructure (alpha-helical structure).

“Obtaining” is understood herein as manufacturing, purchasing, orotherwise coming into possession of.

As used herein, “operably linked” is understood as joined, preferably bya covalent linkage, e.g., joining an amino-terminus of one peptide to acarboxy terminus of another peptide, in a manner that the two or morecomponents that are operably linked either retain their originalactivity, or gain an activity upon joining such that the activity of theoperably linked portions can be assayed and have detectable activityusing at least one of the methods provided in the examples.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. For example,pharmaceutically acceptable carriers for administration of cellstypically is a carrier acceptable for delivery by injection, and do notinclude agents such as detergents or other compounds that could damagethe cells to be delivered. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, subcutaneous, buccal, sublingual, inhaled,intramuscular, intraperotineal, rectal, vaginal and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient that can be combined with acarrier material to produce a single dosage form will generally be thatamount of the compound that produces a therapeutic effect.

As used herein, “plurality” is understood to mean more than one. Forexample, a plurality refers to at least two, three, four, five, or more.

As used herein, the terms “peptide”, “peptide compound” and “peptidicstructure” are intended to include peptides comprised ofnaturally-occurring L-amino acids, as well as peptide derivatives,peptide analogues and peptide mimetics of the naturally-occurringL-amino acid structures. The terms “peptide analogue”, “peptidederivative” and “peptidomimetic” as used herein are intended to includemolecules which mimic the chemical structure of a peptide and retain thefunctional properties of the peptide (e.g., the ability to bindGLP-1-receptor). Approaches to designing peptide analogues, derivativesand mimetics are known in the art. For example, see Farmer, P. S. inDrug Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10,pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition.3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem.24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270.

A “polypeptide” as used herein is understood as two or moreindependently selected natural or non-natural amino acids joined by acovalent bond (e.g., a peptide bond). A polypeptide can include 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or morenatural or non-natural amino acids joined by peptide bonds. Polypeptidesas described herein include full length proteins (e.g., fully processedproteins) as well as shorter amino acids sequences (e.g., fragments ofnaturally occurring proteins or synthetic polypeptide fragments).Examples of the polypeptides of the invention include the hydrophobicstapled insulinotropic polypeptides described herein, and any fragments,analogues, or mimetics thereof.

As used herein, “prevention” is understood as to limit, reduce the rateor degree of onset, or inhibit the development of at least one sign orsymptom of a disease or condition. Prevention can require theadministration of more than one dose of an agent or therapeutic.

A “sample” as used herein refers to a biological material that isisolated from its environment (e.g., blood or tissue from an animal,cells, or conditioned media from tissue culture) and is suspected ofcontaining, or known to contain an analyte, such as a virus, anantibody, or a product from a reporter construct, or, as in thisinvention, measurable levels of the delivered stapled peptide or bloodlevels of a functional read-out analyte such as insulin and/or glucose.A sample can also be a partially purified fraction of a tissue or bodilyfluid. A reference sample can be a “normal” sample, from a donor nothaving the disease or condition fluid, or from a normal tissue in asubject having the disease or condition (e.g., non-infected tissue vs. ainfected tissue). A reference sample can also be from an untreated donoror cell culture not treated with an active agent (e.g., no treatment oradministration of vehicle only). A reference sample can also be taken ata “zero time point” prior to contacting the cell or subject with theagent to be tested.

“Similarity” or “percent similarity” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residues,or conservative substitutions thereof, that are the same when comparedand aligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms, or by visual inspection.

The term “stable” or “stabilized”, as used herein with reference to apolypeptide, refers to polypeptides which have been hydrocarbon-stapledto promote and/or maintain helical structure and/or improve proteaseresistance and/or improve acid stability and/or improve thermalstability and/or improve pharmacologic properties. Stabilizedpolypeptides are a type of structurally constrained polypeptides.Polypeptides may be singly, doubly, triply, or multiply stapled with thesame or different hydrocarbon staple, in accordance with the methodsdescribed herein.

As used herein, “structurally constrained polypeptides” (or“structurally fortified”) and the like are understood to includemodified polypeptides having any (i.e., at least one) chemicalmodification, e.g., mutation of the original or native sequence with anatural or non-natural amino acid; chemical modification to incorporatea molecular tether; chemical modification to promote the formation of adisulfide bridge; etc. such that the structurally constrained peptideadopts a more limited number of structures than the unmodified peptide.A structurally constrained polypeptide can include 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or more mutations as compared to the native, wild-typesequence. For example, molecular tethers can include hydrocarbon staplesto promote the formation of stable helical structures, especiallyalpha-helical and 3₁₀ structures, or kinks depending on the positions ofthe ends of the tethers and the lengths of the tethers. Natural ornon-natural amino acids can be employed to promote kinks (e.g. bends inthe structure as defined by the variable angles between the twoadjoining structures) or other preferred confirmations. For example, thenatural amino acid proline can induce a kink in a peptide due to thestructure of the amino acid R group and the lack of a hydrogen-bonddonor. Non-natural amino acids, particularly those having large and/orcharged R groups, or N-methylated amides, N-substituted glycines, cyclicalpha,alpha-disubstitution, cyclic N,N-disubstitution, and beta-aminoacids can promote specific, desired confirmations. It is understood thata population of “structurally constrained” peptides in solution may notall have the desired confirmation all of the time. Instead, in apopulation of structurally constrained peptides in solution, the desiredconfirmation is present at least about 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, or more of the time than the native or original peptidesequence in solution prior to chemical modification. The structure of apopulation of peptides in solution can be determined by various methodsknown to those of skill in the art including, but not limited to,circular dichroism and NMR spectroscopy. Xray crystallography can beapplied to determine the structure of a constrained peptide when packedin the form of a crystal.

“Small molecule” as used herein is understood as a compound, typicallyan organic compound, having a molecular weight of no more than about1500 Da, 1000 Da, 750 Da, or 500 Da. In an embodiment, a small moleculedoes not include a polypeptide or nucleic acid including only naturalamino acids and/or nucleotides.

An agent, antibody, polypeptide, nucleic acid, or other compound“specifically binds” a target molecule, e.g., antigen, polypeptide,nucleic acid, or other compound, when the target molecule is bound withat least 100-fold, preferably at least 500-fold, preferably at least1000-fold, preferably at least a 5000-fold, preferably at least a10,000-fold preference as compared to a non-specific compounds, or apool of non-specific compounds. Specifically binds can be used inrelation to binding one of two or more related compounds that havephysically related structures. Binding preferences and affinities,absolute or relative, can be determined, for example by determining theaffinity for each pair separately or by the use of competition assays orother methods well known to those of skill in the art.

A “subject” as used herein refers to living organisms. In certainembodiments, the living organism is an animal. In certain preferredembodiments, the subject is a mammal. In certain embodiments, thesubject is a domesticated mammal. Examples of subjects include humans,monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A humansubject may also be referred to as a patient having an abnormal glucosehomeostasis disorder, e.g., type 2 diabetes or at risk of developingtype 2 diabetes.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome has a sufficient number of risk factorsor presents with a sufficient number or combination of signs or symptomsof the disease, condition, or syndrome such that a competent individualwould diagnose or suspect that the subject was suffering from thedisease, condition, or syndrome. Methods for identification of subjectssuffering from or suspected of suffering from conditions such as type 2diabetes is within the ability of those in the art. Subjects sufferingfrom, and suspected of suffering from, a specific disease, condition, orsyndrome are not necessarily two distinct groups.

The pharmaceutical agents may be conveniently administered in unitdosage form and may be prepared by any of the methods well known in thepharmaceutical arts, e.g., as described in Remington's PharmaceuticalSciences (Mack Pub. Co., Easton, Pa., 1985). Formulations for parenteraladministration may contain as common excipients such as sterile water orsaline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. In particular,biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be usefulexcipients to control the release of certain agents. In certainembodiments, orally-deliverable formulations can be prepared based onknown methods, including those described in Remington's PharmaceuticalSciences (Mack Pub. Co., Easton, Pa., 1985), among other known treatiseson pharmaceutical formulations.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to e.g., thespecific compound being utilized, the particular composition formulated,the mode of administration and characteristics of the subject, e.g., thespecies, sex, weight, general health and age of the subject. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

As used herein, “susceptible to” or “prone to” or “predisposed to” aspecific disease or condition and the like refers to an individual whobased on genetic, environmental, health, and/or other risk factors ismore likely to develop a disease or condition than the generalpopulation. An increase in likelihood of developing a disease, e.g.,type 2 diabetes, may be an increase of about 10%, 20%, 50%, 100%, 150%,200%, or more.

Should they appear, the following chemical entities are defined.

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkenyl” refers to aC₂-C₈ alkenyl chain. In the absence of any numerical designation,“alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkynyl” refers to aC₂-C₈ alkynyl chain. In the absence of any numerical designation,“alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Preferred cycloalkyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₀ indicates that the group may have from 1 to10 (inclusive) carbon atoms in it. In the absence of any numericaldesignation, “alkyl” is a chain (straight or branched) having 1 to 20(inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20) carbon atoms in it. The term “alkylene” refers to adivalent alkyl (i.e., —R—).

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. This includes all individual sequences when arange of SEQ ID NOs: is provided. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

The symbol “

”when used as part of a molecular structure refers to a single bond or atrans or cis double bond.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Insulinotropic Polypeptides

Another aspect of the invention provides the insulinotropic polypeptidesof the invention, which can be modified with 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more hydrocarbon staples at any location(s) along the aminosequence of the inventive polypeptides in accordance with the methodsoutlined herein. The invention is not limited to any particularinsulinotropic polypeptide, of a naturally occurring or syntheticsource, and may including any known or yet-to-be identified polypeptidehaving insulinotropic properties, including exendin-4, exenatide,Gastric Inhibitory Polypeptide Preprotein (GIPP), Gastric InhibitoryPeptide (GIP), Glucagon-like Peptide-1 Precursor (GLP-1P), Glucagon-likePeptide-1 (GLP-1), Glucagon-like Peptide-2 (GLP-2), fragments of GLP-1,liraglutide, taspoglutide, albiglutide and LY2189265, all of which areexemplary insulinotropic polypeptides that can be used as templates invarious embodiments of the invention for structural fortification byhydrocarbon stapling (see FIG. 1). The insulinotropic polypeptides canalso include any homolog, analog, derivative, functional fragment ormimetic of any of those polypeptides indicated above.

The alpha helix of any of the insulinotropic polypeptides of theinvention, e.g., exenatide, can be stabilized with at least onehydrocarbon staple using methods for hydrocarbon stapling describedherein or in accordance with the teachings of the methods of U.S.Publication No. 2006/0014675A1, 2006/0008848A1, 2004/0171809A1 and U.S.Pat. Nos. 7,723,469, 7,192,713, and 7,084,244, each of which areincorporated herein by reference. In other embodiments, more than one,including at least 2, 3, or even 4 or even up to 12 or more hydrocarbonstaples can be used to stabilize the exenatide of the invention.Hydrocarbon staples suitable for use with any of the modifiedpolypeptides are described herein and in U.S. Publication No.2005/0250680 or in U.S. Pat. No. 7,723,469 (“Stabilized alpha helicalpeptides and uses thereof”), each of which are incorporated by referencein their entireties. Hydrocarbon stapling allows a polypeptide,predisposed to have a helical secondary structure, to maintain itsnative helical conformation and increase its stability and efficacy. Inone embodiment, the modified polypeptide has at least 10%, 20%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or more helicity in an aqueoussolution as determined by circular dichroism. Assays for determiningcircular dichroism are known in the art and described herein.

In a particular embodiment, an insulinotropic polypeptide, e.g.,exenatide, of the invention can include one hydrocarbon staple near theN-terminus. In another particular embodiment, an insulinotropicpolypeptide, e.g., exenatide, of the invention can include onehydrocarbon staple near the C-terminus. In another particularembodiment, the exenatide of the invention can include a hydrocarbonstaple in the middle of the sequence and at any position between the N-and C-terminii. In yet another embodiment, the exenatide of theinvention can include one hydrocarbon staple near the C-terminus andanother hydrocarbon staple near the N-terminus. In yet anotherembodiment, the exenatide of the invention can include one hydrocarbonstaple near the C-terminus and another hydrocarbon staple in the middleof the sequence, or one hydrocarbon stapled near the N-terminus andanother in the middle of the peptide sequence.

In yet another embodiment, the insulinotropic polypeptides of theinvention may be sequentially truncated from either the N-terminus orthe C-terminus, or in an alternating fashion from the N- and C-terminiiwith hydrocarbon staples being inserted into the foreshortened variants.

Exemplary stapled polypeptides of the invention are shown in FIG. 7.

The hydrocarbon stapled polypeptides include a tether (linkage) betweentwo amino acids, which tether significantly enhances the helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., about 3.4 or about7 amino acids). Accordingly, amino acids positioned at i and i+3; i andi+4; or i and i+7 are ideal candidates for chemical modification andcross-linking. Thus, any of the amino acid residues of the modifiedpolypeptides of the invention may be tethered (e.g., cross-linked) inconformity with the above. Suitable tethers are described herein and inU.S. Patent Publication No. 2005/0250680 and U.S. Pat. No. 7,723,469(both of which are incorporated herein by reference). It is understoodthat tethers such as hydrocarbon staples can be positioned at otherintervals to promote helical variants (e.g. with different pitches,angles, or residues and fractions thereof per turn) or structures otherthan helices.

In a further embodiment, the hydrocarbon staple(s) is positioned so asto link a first amino acid (i) and a second amino acid (i+3) which is 3amino acids downstream of the first amino acid. In another embodiment,the hydrocarbon staple links a first amino acid (i) and a second aminoacid (i+4) which is 4 amino acids downstream of the first amino acid. Inyet another embodiment, the hydrocarbon staple links a first amino acid(i) and a second amino acid (i+7) which is 7 amino acids downstream ofthe first amino acid.

Mutations, Truncations, and Extensions of Insulinotropic Polypeptides

The invention contemplates insulinotropic peptides (e.g., exenatide)having conserved and non-conserved amino acid substitutions. Conservedamino acid substitutions consist of replacing one or more amino acidswith amino acids of similar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to asparticacid (D), aspartic acid (D) to asparagine (N), and glutamic acid (E) toglutamine (Q) amino acid substitution. Non-conserved substitutionsconsist of replacing one or more amino acids with amino acids possessingdissimilar charge, size, and/or hydrophobicity characteristics, such as,for example, a glutamic acid (E) to valine (V) substitution.Substitutions can include the use of conserved or non-conservednon-natural amino acids.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the full-length or truncated exenatide peptides,as well as at a position internal to the peptide. Such insertions willgenerally range from 2 to 15 amino acids in length. It is contemplatedthat insertions made at either the carboxy or amino terminus of thepeptide of interest may be of a broader size range, with about 2 toabout 50 amino acids being preferred. One or more such insertions may beintroduced into full-length or truncated insulinotropic polypeptides(e.g., exenatide), as long as such insertions result in modifiedpeptides which may still exhibit at least about 50%, or 60%, or 70%, or80%, or 90%, or 95%, or 99% or higher the activity of a comparablestarting polypeptide (e.g., modified exenatide of SEQ ID NO: 1). Suchchanges preferably also result in exenatide variants that remain orallybioavailable and/or retain prolonged stability in the blood and tissues.

Deletions of full-length or truncated exenatide peptides are also withinthe scope of the invention. Such deletions consist of the removal of oneor more amino acids from the exenatide peptides; or exenatide variants,with the lower limit length of the resulting peptide sequence being 4,5, or 6 amino acids. Such deletions may involve a single contiguous orgreater than one discrete portion of the peptide sequences. One or moresuch deletions may be introduced into full-length or truncatedinsulinotropic peptides (e.g., exenatide), as long as such deletionsresult in peptides which may still exhibit at least about 50%, or 60%,or 70%, or 80%, or 90%, or 95%, or 99% or higher the activity of acomparable starting polypeptide (e.g., modified exenatide of SEQ ID NO:1). and which are preferably orally bioavailable and/or retain prolongedstability in the blood and tissues.

Stabilization of Insulinotropic Polypeptides (e.g., Exenatide)

The modified insulinotropic polypeptides (e.g., exenatide) of thepresent invention are structurally constrained (e.g., stabilized,stapled) helical and/or include one or more amino acid sequencemodifications as compared to the native (i.e., wild type or otherwisenaturally occurring) sequence to incorporate natural and/or non-naturalamino acids to limit the structural flexibility of the peptide ascompared to the native sequence, which can lose bioactive shape whentaken out of physiologic context. Preferably, the insulinotropicpolypeptides of the invention include at least one molecular tether suchas a hydrocarbon staple. Hydrocarbon stapling is described in U.S.Patent Publication No. 2005/0250680 or U.S. Pat. No. 7,723,469, whichare incorporated herein by reference in their entirety, as well asWalensky et al. Science, 2004, 305: 1466-70; Walensky et al. Mol. Cell.2006, October 20; 24(2):199-210; Bernal et al. J Am Chem. Soc. 2007 Apr.25; 129(16):5298; Danial et al Nat Med 2008, 2008 February;14(2):144-53. Epub 2008 Jan. 27; Gavathiotis et al Nature 2008, October23; 455(7216):1047-9; Stewart et al. Nature Chem Bio 2010 Jun. 20(electronically), as well as in U.S. Publication No. 2006/0014675A1,2006/0008848A1, 2004/0171809A1 and U.S. Pat. Nos. 7,192,713, and7,084,244, each of which are incorporated herein by reference, all ofwhich are incorporated herein by reference.

The peptide α-helix participates in critically important proteininteractions by presenting specific amino acid residues in an orderedand precise arrangement over a relatively large contact surface area(Chittenden, T., et al., Embo Journal, 1995. 14(22): p. 5589-5596;Kussie, P. H., et al. Science, 1996. 274(5289): p. 948-953; Ellenberger,T. E., et al., Cell, 1992. 71(7): p. 1223-1237). Alpha-helical domainsand other protein structural features are frequently stabilized byscaffold sequences in the remainder of the protein, which facilitate theformation and/or maintenance of a helical structure, e.g., an α-helicalstructure. When taken out of context, α-helical peptide motifs canunfold, leading to loss of biological activity. Critical challenges isdeveloping α-helical peptides include promoting and/or maintaining theirnatural α-helical structure and preparing peptides that can resistproteolytic, acid and thermal degradation, and thereby remain intact invivo.

For example a structurally constrained peptide of the invention caninclude an alpha-helical domain of an exenatide polypeptide. In someinstances, the structurally constrained peptide is constrained with ahydrocarbon tether at the N-terminus. In some instances, thestructurally constrained peptide is constrained with a hydrocarbontether at the C-terminus. In other instances, the structurallyconstrained peptide is constrained with a hydrocarbon tether within themiddle of the peptide sequence. In still further instances, thestructurally constrained peptide is constrained with more than onehydrocarbon tether, e.g., wherein at least one or both of the tethers isat or near a terminus of the peptide (i.e., beginning at 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 residues from the N- or C-terminusof the peptide). In a particular embodiment, the exenatide peptidecomprises a tether at or near both the N- and C-terminii (i.e.,beginning at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15residues from the N- or C-terminus of the peptide). These particularpositions for hydrocarbon stapling are not meant to be limiting in anyway as placement of the hydrocarbon staple (multiple staples) can be atany location (or combination of locations) along the length of a giveninsulinotropic polypeptide. Such positions can be determined andoptimized empirically and without undue experimentation utilizing thestaple scanning methods described and shown in the figures of thisspecification.

The hydrocarbon stapled polypeptides include a tether (linkage) betweentwo amino acids, which tether significantly enhances the helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., about 3-3.6 orabout 7 amino acids). Accordingly, amino acids positioned at i and i+3;i and i+4; or i and i+7 are ideal candidates for chemical modificationand cross-linking. Thus, for example, where a peptide has the sequence .. . X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , cross-links between X1and X4, or between X1 and X5, or between X1 and X8 are useful as arecross-links between X2 and X5, or between X2 and X6, or between X2 andX9, etc. The use of multiple cross-links (e.g., 2, 3, 4 or more) hasalso been achieved, compounding the benefits of individual stapledadducts (e.g. improved helicity and activity; improved helicity andthermal stability; improved helicity and acid stability; improvedhelicity and pharmacologic properties). Thus, the invention encompassesthe incorporation of more than one crosslink within the polypeptidesequence to either further stabilize the sequence or facilitate thestructural stabilization, proteolytic resistance, thermal stability,acid stability, pharmacologic properties, and biological activityenhancement of longer polypeptide stretches. The invention alsocontemplates that a pair of sequentially-occurring staples or tethersmay be formed in a “stitched” configuration, i.e., wherein the end of afirst-occurring tether originates from the same residue as the beginningof a second sequentially-occurring staple, e.g., as exemplified in FIG.5B.

In some embodiments of the invention, the tethers, e.g., hydrocarbonstaples are used to stabilize structures other than helices. In suchcases, the ends of the tethers can be placed at intervals other than ati, i+3, i+4, and i+7. For example, a molecular tether can be used tostabilize the kink region of a peptide domain to produce a curvedsurface rather than a flat continuous face as with a helix. The aminoacid sequence and the placement of the ends of the tether will determinethe number of amino acids spanned by the tether. Such considerations arewell understood by those of skill in the art.

In one embodiment, the modified polypeptides of the invention have theformula (I),

wherein;each R₁ and R₂ are independently H or a C₁ to C₁₀ alkyl, alkenyl,alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl;R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n); each of which issubstituted with 0-6 R₅;R₄ is alkyl, alkenyl, or alkynyl;R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;n is an integer from 1-4;x is an integer from 2-10;each y is independently an integer from 0-100;z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); andeach Xaa is independently an amino acid. The modified polypeptides mayincludes an amino acid sequence which forms an alpha-helix and is 30% ormore identical to, or contain at least 7 contigous amino acids from anamino acid sequence of SEQ ID NO:1-4, wherein X is any amino acid andfurther identifies the amino acid residues which are linked by ahydrocarbon staple, and B is methionine or norleucine.

The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., C₅,C₈ or C₁₁ alkyl or a C₅, C₈ or C₁₁ alkenyl, or C₅, C₈ or C₁₁ alkynyl).The tethered amino acid can be alpha disubstituted (e.g., C₁-C₃ ormethyl).

In some instances, x is 2, 3, or 6.

In some instances, each y is independently an integer between 3 and 15.

In some instances each y is independently an integer between 1 and 15.

In some instances, R₁ and R₂ are each independently H or C₁-C₆alkyl.

In some instances, R₁ and R₂ are each independently C₁-C₃ alkyl.

In some instances, at least one of R₁ and R₂ are methyl. For example R₁and R₂ are both methyl.

In some instances R₃ is alkyl (e.g., C₈ alkyl) and x is 3.

In some instances, R₃ is C₁₁ alkyl and x is 6.

In some instances, R₃ is alkenyl (e.g., C₈ alkenyl) and x is 3.

In some instances x is 6 and R₃ is C′₁₁ alkenyl.

In some instances, R₃ is a straight chain alkyl, alkenyl, or alkynyl.

In some instances R₃ is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.

In certain embodiments the two alpha, alpha disubstituted stereocentersare both in the R configuration or S configuration (e.g., i, i+4cross-link), or one stereocenter is R and the other is S (e.g., i, i+7cross-link). Thus, where formula I is depicted as

the C′ and C″ disubstituted stereocenters can both be in the Rconfiguration or they can both be in the S configuration, for examplewhen X is 3. When x is 6, the C′ disubstituted stereocenter is in the Rconfiguration and the C″ disubstituted stereocenter is in the Sconfiguration. The R₃ double bond may be in the E or Z stereochemicalconfiguration.

In some instances R₃ is [R₄—K—R₄]_(n); and R₄ is a straight chain alkyl,alkenyl, or alkynyl.

In some embodiments the modified polypeptide comprises at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,or more contiguous amino acids of an insulinotropic polypeptide. Each[Xaa]_(y) is a peptide that can independently comprise at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more contiguousamino acids of a insulinotropic polypeptide, e.g., the sequences of FIG.1.

The modified polypeptide can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 contiguous aminoacids of an insulinotropic polypeptide, e.g., the sequences of FIG. 1,wherein two amino acids that are separated by two, three, or six aminoacids are replaced by amino acid substitutes that are linked via R₃.Thus, at least two amino acids can be replaced by tethered amino acidsor tethered amino acid substitutes. Thus, where formula (I) is depictedas

[Xaa]_(y)′, and [Xaa]_(y)″ can each comprise contiguous polypeptidesequences from the same or different heptad repeat or heptad repeat likedomains.

The invention features cross-linked polypeptides comprising 10 (11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50or more) contiguous amino acids of an insulinotropic polypeptide, e.g.,the sequences of FIG. 1, wherein the alpha carbons of two amino acidsthat are separated by two, three, or six amino acids are linked via R₃,one of the two alpha carbons is substituted by R₁ and the other issubstituted by R₂ and each is linked via peptide bonds to additionalamino acids.

In another embodiment, the modified polypeptides of the invention havethe formula (II),

whereineach R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl; heteroarylalkyl; or heterocyclylalkyl;each n is independently an integer from 1-15;x is 2, 3, or 6each y is independently an integer from 0-100;z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);each Xaa is independently an amino acid.

The modified polypeptide forms an alpha-helix and can have an amino acidsequence which forms an alpha-helix and is 30% or more identical to, orcontain at least 7 contiguous amino acids from an amino acid sequence ofSEQ ID NOs: 2, 15-38 (exenatide) and 39-62 (GLP1(7-37)) wherein X is anyamino acid and further identifies the amino acid residues which arelinked by a hydrocarbon staple, and B is methionine or norleucine. Themodified polypeptides may include an amino acid sequence that forms analpha-helix and is 30% or more identical to, or contain at least 3,preferably at least 7 contigous amino acids from an amino acid sequence,or at least two amino acids from a face of a helix formed by a peptidehaving the sequence of SEQ ID NOs: 2, 15-38 (exenatide) and 39-62(GLP1(7-37)) wherein X is any amino acid and further identifies theamino acid residues which are linked by a hydrocarbon staple, and B ismethionine or norleucine.

In still another embodiment, the modified polypeptides of the inventionhave the formula (III),

wherein;each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or a naturally occurringamino acid side chain; each of which is substituted with 0-6 R₅;R₄ is alkyl, alkenyl, or alkynyl;R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;R₇ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or an naturally occurringamino acid side chain; each of which is substituted with 0-6 R₅;n is an integer from 1-4;x is an integer from 2-10;each y is independently an integer from 0-100;z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); andeach Xaa is independently an amino acid.

The modified polypeptides may include an amino acid sequence that formsan alpha-helix and is 30% or more identical to, or contain at least 7contigous amino acids from an amino acid sequence, or at least two aminoacids from a face of a helix formed by a peptide having the sequence ofSEQ ID NOs: 2, 15-38 (exenatide) and 39-62 (GLP1(7-37)), wherein X isany amino acid and further identifies the amino acid residues which arelinked by a hydrocarbon staple, and B is methionine or norleucine.

While hydrocarbon tethers have been described, other tethers are alsoenvisioned. For example, the tether can include one or more of an ether,thioether, ester, amine, or amide moiety. In some cases, a naturallyoccurring amino acid side chain can be incorporated into the tether. Forexample, a tether can be coupled with a functional group such as thehydroxyl in serine, the thiol in cysteine, the primary amine in lysine,the acid in aspartate or glutamate, or the amide in asparagine orglutamine. Accordingly, it is possible to create a tether usingnaturally occurring amino acids rather than using a tether that is madeby coupling two non-naturally occurring amino acids. It is also possibleto use a single non-naturally occurring amino acid together with anaturally occurring amino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary structure, whereas, in some instances, it is desirable toprovide less constraint on the secondary structure, and thus a longertether may be desired. It is further understood that the insertion of atether at a site or in an amino acid sequence when the amino acidsequence has no tendency to form a helix will not result in helixformation.

Additionally, while examples of tethers spanning from amino acids i toi+3, i to i+4; and i to i+7 have been described in order to provide atether that is primarily on a single face of the alpha helix, thetethers can be synthesized to span any combinations of numbers of aminoacids to promote and/or maintain the structures other than alphahelices.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the described herein will be evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof. The specific method ofsynthesis of the peptides is not a limitation of the invention.

Synthesis of the Polypeptides of the Invention

The peptides of this invention can be made by chemical synthesismethods, which are well known to the skilled artisan and describedherein. See, for example, Fields et al., Chapter 3 in SyntheticPeptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York,N.Y., 1992, p. 77. Hence, peptides can be synthesized using theautomated Merrifield techniques of solid phase synthesis with thealpha-NH₂ protected by either t-Boc or F-moc chemistry using side chainprotected amino acids on, for example, an Applied Biosystems PeptideSynthesizer Model 430A or 431 or the AAPPTEC multichannel synthesizerAPEX 396.

One manner of making of the peptides described herein is using solidphase peptide synthesis (SPPS). The C-terminal amino acid is attached toa cross-linked polystyrene resin via an acid labile bond with a linkermolecule. This resin is insoluble in the solvents used for synthesis,making it relatively simple and fast to wash away excess reagents andby-products. The N-terminus is protected with the Fmoc group, which isstable in acid, but removable by base. Any side chain functional groupsare protected with base stable, acid labile groups.

Longer peptides can also be made by conjoining individual syntheticpeptides using native chemical ligation. Alternatively, longer syntheticpeptides can be synthesized by well known recombinant DNA techniques.Such techniques are provided in well-known standard manuals withdetailed protocols. To construct a coding sequence encoding a peptide ofthis invention, the amino acid sequence is reverse translated to obtaina nucleic acid sequence encoding the amino acid sequence, preferablywith codons that are optimum for the organism in which the gene is to beexpressed. Next, a coding sequence is made, typically by synthesizingoligonucleotides which encode the peptide and any regulatory elements,if necessary. The coding sequence is inserted in a suitable cloningvector and transfected into a host cell. Furthermore, the host cell isengineered so as to be able to incorporate the non-natural amino acidsfor the hydrocarbon staple. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. SeeLiu et al. Proc. Nat. Acad. Sci. (USA), 94:10092-10097 (1997). Thepeptide is purified and characterized by standard methods.

The peptides can be made in a high-throughput, combinatorial fashion,e.g., using a high-throughput polychannel combinatorial synthesizer suchas that available from Advanced Chemtech/APPTTEC.

Assaying Activity of the Insulinotropic Polypeptides of the Invention

Described herein, are methods for evaluating the ability of astructurally constrained insulinotropic polypeptide of the invention(e.g., exenatide) to enhance the production of insulin, either in vitro,in vivo, or preferably both. Specifically, such assays are describedbelow and in the Examples. Additional assays for evaluating theinsulin-enhancing activity of exenatide are well known to those withordinary skill in the art. In addition, described herein, are methodsfor evaluating other aspects and properties of the insulinotropicpolypeptides of the invention (e.g., exenatide), including theirimproved bioavailability when delivered orally, and other benefits ofthe polypeptides of the invention, including, but are not limited to,their extended half-life, their enhanced alpha-helicity, their improvedthermal stability and their protease resistance, and their increasedfunctional activity and pharmacologic properties, improvedbioavailability when administered by any route. Many of these propertiesare described and demonstrated in the Examples; however, it is notedthat one or ordinary skill in the art will be able to ascertain withoutundue experimentation the improved properties imparted on theinsulinotropic polypeptides of the invention using known methods andassays. For example, assays can include protease resistance assays,plasma stability assays, pharmacokinetics assays, GLP-1-receptor bindingassays, GLP-1-receptor second messenger signaling assays,glucose-stimulated insulin release cellular assays, in vivo monitoringof serum glucose and insulin levels in response to insulinotropicpeptide treatment, and general ELISA or antibody-based methods formeasuring quantities of insulin, glucagon or other glucoregulatorycomponent in the blood, all of which will be understood in the contextof the herein Examples, as well as based on the knowledge possessed byone of ordinary skill in the art. All of such assays and measurementscan be performed without undue experimentation.

In a particular example, insulinotropic activity can be measured bydetection of insulin in a sample. The present invention concernsmodified insulinotropic polypeptides that exceeds or equals theinsulinotropic activity of the non-modified insulinotropic polypeptides.The insulinotropic property of an agent may be determined by providingthat agent to animal cells, or injecting that agent into animals andmonitoring the release of immunoreactive insulin (IRI) into the media orcirculatory system of the animal, respectively. The presence of IRI isdetected through the use of a radioimmunoassay which can specificallydetect insulin.

Although any radioimmunoassay capable of detecting the presence of IRImay be employed, one specific method is to use a modification of theassay method of Albano, J. D. M., et al., (Acta Endocrinol. 70:487-509(1972)). In this modification, a phosphate/albumin buffer with a pH of7.4 is employed. The incubation is prepared with the consecutivecondition of 500 microliters of phosphate buffer, 50 microliters ofperfusate sample or rat insulin standard in perfusate, 100 microlitersof anti-insulin antiserum (Wellcome Laboratories; 1:40,000 dilution),and 100 microliters of [¹²⁵I] insulin, giving a total volume of 750microliters in a 10×75-mm disposable glass tube. After incubation for2-3 days at 4° C., free insulin is separated from antibody-bound insulinby charcoal separation. The assay sensitivity is generally 1-2microliters U/ml. In order to measure the release of IRI into the cellculture medium of cells grown in tissue culture, one preferablyincorporates radioactive label into proinsulin. Although any radioactivelabel capable of labeling a polypeptide can be used, it is preferable touse 3 H leucine in order to obtain labeling of proinsulin. Labeling canbe done for any period of time sufficient to permit the formation of adetectably labeled pool of proinsulin molecules; however, it ispreferable to incubate cells in the presence of radioactive label for a60-minute time period. Although any cell line capable of expressinginsulin can be used for determining whether a compound has aninsulinotropic effect, it is preferable to use rat insulinoma cells, andespecially RIN-38 rat insulinoma cells. Such cells can be grown in anysuitable medium; however, it is preferable to use DME medium containing0.1% BSA and 25 mM glucose.

The insulinotropic property of a hydrocarbon stapled polypeptide of theinvention may also be determined by pancreatic infusion. The in situisolated perfused rat pancreas preparation is a modification of themethod of Penhos, J. C., et al. (Diabetes 18:733-738 (1969)). Inaccordance with such a method, fasted rats (preferably male CharlesRiver strain albino rats), weighing 350-600 g, are anesthetized with anintraperitoneal injection of Amytal Sodium (Eli Lilly and Co., 160ng/kg). Renal, adrenal, gastric, and lower colonic blood vessels areligated. The entire intestine is resected except for about four cm ofduodenum and the descending colon and rectum. Therefore, only a smallpart of the intestine is perfused, thus minimizing possible interferenceby enteric substances with insulinotropic immunoreactivity. Theperfusate can be a modified Krebs-Ringer bicarbonate buffer with 4%dextran T70 and 0.2% bovine serum albumin (fraction V), and can bebubbled with 95% O₂ and 5% CO₂. A nonpulsatile flow, four-channelroller-bearing pump (Buchler polystatic, Buchler Instruments Division,Nuclear-Chicago Corp.) can be used, and a switch from one perfusatesource to another can be accomplished by switching a three-way stopcock.The manner in which perfusion is performed, modified, and analyzed canfollow the methods of Weir, G. C., et al., (J. Clin. Investigat.54:1403-1412 (1974)), which are hereby incorporated by reference.

Pharmaceutical Compositions and Routes of Administration

One or more structurally constrained insulinotropic polypeptides of theinstant invention can be used in a pharmaceutical composition for theprevention and/or treatment of type 2 diabetes and related disorders.Combinations of pharmaceutical agents are frequently used for thetreatment of complex or dangerous diseases, such as diabetes. Treatmentand prevention method provided herein can be performed using acombination of the structurally constrained insulinotropic polypeptides,which can be selected and combined to prevent the development ofresistance, or can be selected and combined depending on the status andnature of the disorder in the subject. A pharmaceutical composition ofthe instant invention can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or more structurally constrained insulinotropicpolypeptides. The structurally constrained peptides can also be combinedwith other agents, e.g., known treatments or therapies for treatingdiabetes.

As used herein, the compounds of this invention are defined to includepharmaceutically acceptable derivatives thereof. A “pharmaceuticallyacceptable derivative” means any pharmaceutically acceptable salt,ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives are those that increase thebioavailability of the compounds of this invention when such compoundsare administered to a mammal (e.g., by allowing an orally administeredcompound to be more readily absorbed into the blood, to increase serumstability or decrease clearance rate of the compound) or which enhancedelivery of the parent compound to a biological compartment (e.g., thebrain or lymphatic system) relative to the parent species. Derivativesinclude derivatives where a group which enhances aqueous solubility oractive transport through the gut membrane is appended to the structureof formulae described herein.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system, pancreas), increase oralavailability, increase solubility to allow administration by injection,alter metabolism and alter rate of excretion. Pharmaceuticallyacceptable salts of the compounds of this invention include thosederived from pharmaceutically acceptable inorganic and organic acids andbases. Examples of suitable acid salts include acetate, adipate,benzoate, benzenesulfonate, butyrate, citrate, digluconate,dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,palmoate, phosphate, picrate, pivalate, propionate, salicylate,succinate, sulfate, tartrate, tosylate and undecanoate. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium), alkalineearth metal (e.g., magnesium), ammonium and N-(alkyl)₄₊ salts. Thisinvention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

The compounds of the invention can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, intravaginally, cervically,topically, in an ophthalmic preparation, or by inhalation, with a dosageranging from about 0.001 to about 100 mg/kg of body weight, or accordingto the requirements of the particular drug and more preferably from0.5-10 mg/kg of body weight. The methods herein contemplateadministration of an effective amount of compound or compoundcomposition to achieve the desired or stated effect.

In a preferred composition, the insulinotropic polypeptides of theinvention are formulated for oral delivery. Any suitablepharmaceutically acceptable excipients, carriers, solvents, stabilizers,etc. can be used for obtaining an oral formulation of the exenatidepeptides of the invention. Oral delivery may be by any suitable means,including by tablet, pill, suspension, quick-release strips, etc.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 1% to about 95% active compound(w/w). Alternatively, such preparations contain from about 20% to about80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise a compound of theinvention or a pharmaceutically acceptable salt thereof; an additionalagent including for example, one or more therapeutic agents for theprevention and/or treatment of diabetes, including, but not limited to,insulin, sulfonylureas (tolbutamide (Orinase), acetohexamide (Dymelor),tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide(Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride(Amaryl), and gliclazide (Diamicron)), meglitinides (repaglinide(Prandin), nateglinide (Starlix)), Biguanides (metformin, phenformin,buformin), Thiazolidinediones, Alpha-glucosidase inhibitors, DPP-4inhibitors, non-stapled Incretin mimetics, and Amylin analogues.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α.-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tween® or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropyle- ne-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin,may also be advantageously used to enhance delivery of compounds of theformulae described herein.

The pharmaceutical compositions of this invention may be administeredenterally for example by oral administration, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir, preferably by oral or vaginal administrationor administration by injection. The pharmaceutical compositions of thisinvention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases, or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, and intracranial injection orinfusion techniques.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a patient,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween® 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

The pharmaceutical compositions of the invention may be administeredtopically or intravaginally. The pharmaceutical composition will beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. In still another embodiment, the pharmaceuticalcomposition is formulated as a vaginal ring. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topically-transdermalpatches and iontophoretic administration are also included in thisinvention. In one embodiment, the compound of the invention isadministered vaginally as a prophylactic treatment for a sexuallytransmitted disease.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

Effective dosages of the peptides or antibodies targeted hereto of theinvention to be administered may be determined through procedures wellknown to those in the art which address such parameters as biologicalhalf-life, bioavailability, and toxicity.

A therapeutically effective dose refers to that amount of the compoundor antibody sufficient to result in amelioration of symptoms or aprolongation of survival in a patient.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with abnormal or aberrant glucosehomeostasis, including, for example, insulin-dependent diabetes mellitus(type 1 diabetes) and noninsulin-dependent diabetes mellitus (type 2diabetes). As used herein, the term “treatment” is defined as theapplication or administration of a therapeutic agent to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease, a symptom ofdisease or a predisposition toward a disease, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease, the symptoms of disease or the predisposition towarddisease. A therapeutic agent includes, but is not limited to, smallmolecules, peptides, antibodies, ribozymes and antisenseoligonucleotides.

The polypeptides of the present invention may also be employed incombination with other pharmaceutical agents useful in the treatment ofdiabetes, impaired fasting glucose, impaired glucose tolerance,hyperglycemia, and obesity. Suitable agents include insulinsecretagogues, insulin sensitizers, and metformin HCl.

The present invention includes methods for the treatment of diabetes andrelated diseases and conditions. One such method comprises the step ofadministering to a subject in need thereof, a therapeutically effectiveamount of one or more hydrocarbon stapled insulinotropic polypeptides ofthe invention.

Polypeptides of the invention may be used in methods of the invention totreat diseases, such as diabetes, including type 2 diabetes. Suchmethods may also delay the onset of diabetes and diabetic complications.Other diseases and conditions that may be treated or prevented usingpolypeptides of the invention in methods of the invention include:Maturity-Onset Diabetes of the Young (MODY) (Herman, et al., Diabetes43:40 (1994)), Latent Autoimmune Diabetes Adult (LADA) (Zimmet, et al.,Diabetes Med. 11:299 (1994)), impaired glucose tolerance (IGT) (ExpertCommittee on Classification of Diabetes Mellitus, Diabetes Care 22(Supp. 1) S5 (1999)), impaired fasting glucose (IFG) (Charles, et al.,Diabetes 40:796 (1991)), gestational diabetes (Metzger, Diabetes, 40:197(1991), and metabolic syndrome X.

Polypeptides of the invention may also be used in methods of theinvention to treat secondary causes of diabetes (Expert Committee onClassification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1), S5(1999)). Such secondary causes include glucocorticoid excess, growthhormone excess, pheochromocytoma, and drug-induced diabetes. Drugs thatmay induce diabetes include, but are not limited to, pyriminil,nicotinic acid, glucocorticoids, phenyloin, thyroid hormone,.beta.-adrenergic agents, .alpha.-interferon and drugs used to treat HIVinfection.

Polypeptides of this invention may also be useful for the treatment ofbulimia and obesity including associated dyslipidemia and other obesity-and overweight-related complications such as, for example, cholesterolgallstones, cancer (e.g., colon, rectum, prostate, breast, ovary,endometrium, cervix, gallbladder, and bile duct), menstrualabnormalities, infertility, polycystic ovaries, osteoarthritis, andsleep apnea, as well as for a number of other pharmaceutical usesassociated therewith, such as the regulation of appetite and foodintake, dyslipidemia, hypertrigyceridemia, atherosclerotic diseases suchas heart failure, hyperlipidemia, hypercholesteremia, low HDL levels,hypertension, cardiovascular disease (including atherosclerosis,coronary heart disease, coronary artery disease, and hypertension),cerebrovascular disease and peripheral vessel disease. The polypeptidesof this invention may also be useful for treating physiologicaldisorders related to, for example, regulation of insulin sensitivity,inflammatory response, plasma triglycerides, HDL, LDL, and cholesterollevels and the like.

The methods and polypeptides of the present invention may be used aloneor in combination with additional therapies and/or compounds known tothose skilled in the art in the treatment of diabetes and relateddisorders. Alternatively, the methods and polypeptides described hereinmay be used, partially or completely, in combination therapy.

Polypeptides of the invention may also be administered in combinationwith other known therapies for the treatment of diabetes, including PPARagonists, sulfonylurea drugs, non-sulfonylurea secretagogues,alpha-glucosidase inhibitors, insulin sensitizers, insulinsecretagogues, hepatic glucose output lowering compounds, insulin andanti-obesity drugs. Such therapies may be administered prior to,concurrently with or following administration of the compound of theinvention. Insulin includes both long and short acting forms andformulations of insulin. PPAR agonist may include agonists of any of thePPAR subunits or combinations thereof. For example, PPAR agonist mayinclude agonists of PPAR-alpha, PPAR-gamma, PPAR-delta or anycombination of two or three of the subunits of PPAR. Such PPAR agonistsinclude, for example, rosiglitazone and pioglitazone. Sulfonylurea drugsinclude, for example, glyburide, glimepiride, chlorpropamide, andglipizide. Alpha-glucosidase inhibitors that may be useful in treatingdiabetes when administered with a compound of the invention includeacarbose, miglitol and voglibose. Insulin sensitizers that may be usefulin treating diabetes when administered with a compound of formula (I)include thiozolidinediones and non-thiozolidinediones. Hepatic glucoseoutput lowering compounds that may be useful in treating diabetes whenadministered with a compound of the invention include metformin, such asGLUCOPHAGE™ and GLUCOPHAGE XR™ Insulin secretagogues that may be usefulin treating diabetes when administered with a compound of the inventioninclude sulfonylurea and non-sulfonylurea drugs: GLP-1, GIP, PAC/VPACreceptor agonists, secretin, nateglinide, meglitinide, repaglinide,glibenclamide, glimepiride, chlorpropamide, glipizide. GLP-1 includesderivatives of GLP-1 with longer half-lives than native GLP-1, such as,for example, fatty-acid derivatized GLP-1 and exendin. In one embodimentof the invention, polypeptides of the invention are used in combinationwith insulin secretagogues to increase the sensitivity of pancreaticbeta cells to the insulin secretagogue.

Polypeptides of the invention may also be used in methods of theinvention in combination with anti-obesity drugs. Anti-obesity drugsinclude beta-3 agonists, CB-1 antagonists, appetite suppressants, suchas, for example, sibutramine (MERIDIA™), and lipase inhibitors, such as,for example, orlistat (XENICAL™).

Polypeptides of the invention may also be used in methods of theinvention in combination with drugs commonly used to treat lipiddisorders in diabetic patients. Such drugs include, but are not limitedto, HMG-CoA reductase inhibitors, nicotinic acid, bile acidsequestrants, and fibric acid derivatives. Polypeptides of the inventionmay also be used in combination with anti-hypertensive drugs, such as,for example, β-blockers and ACE inhibitors.

Such co-therapies may be administered in any combination of two or moredrugs (e.g., a compound of the invention in combination with an insulinsensitizer and an anti-obesity drug). Such co-therapies may beadministered in the form of pharmaceutical compositions, as describedabove.

Kits

The present invention also encompasses a finished packaged and labeledpharmaceutical product or laboratory reagent. This article ofmanufacture includes the appropriate instructions for use in anappropriate vessel or container such as a glass vial or other containerthat is hermetically sealed. A pharmaceutical product may contain, forexample, a compound of the invention in a unit dosage form in a firstcontainer, and in a second container, sterile water or adjuvant forinjection. Alternatively, the unit dosage form may be a solid suitablefor oral, transdermal, intranasal, intravaginal, cervical ring, ortopical delivery.

In a specific embodiment, the unit dosage form is suitable forintravenous, intramuscular, intraperiteneal, intranasal, oral,intravaginal, cervical, topical or subcutaneous delivery. Thus, theinvention encompasses solutions, solids, foams, gels, preferablysterile, suitable for each delivery route.

In another specific embodiment, the unit dosage form is suitable fororal administration. Thus, the invention encompasses solutions, solids,foams, gels, preferably sterile, suitable for the oral route.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician, or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instructions indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures (e.g. detection and quantitation of infection), and othermonitoring information.

Specifically, the invention provides an article of manufacture includingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of a pharmaceutical agent contained withinsaid packaging material, wherein said pharmaceutical agent comprises acompound of the invention, and wherein said packaging material includesinstruction means which indicate that said compound can be used toprevent, manage, treat, and/or ameliorate one or more symptomsassociated with a disease, or to stimulate an immune response to preventa disease by administering specific doses and using specific dosingregimens as described herein.

The following examples are provided merely as illustrative of variousaspects of the invention and shall not be construed to limit theinvention in any way.

EXAMPLES Example 1 Materials and Methods Synthesis of HydrocarbonStapled Alpha Helical Polypeptides.

A combined strategy of structural analysis and chemical synthesis wasapplied to construct the modified, structurally constrained peptides.Asymmetric syntheses of (R)-Fmoc-(2′-pentenyl)alanine (“R5”),(S)-Fmoc-(2′-pentenyl)alanine (“S5”), (R)-Fmoc-(2′-octenyl)alanine(“R8”), (S)-Fmoc-(2′-octenyl)alanine (“R8”) α,α-disubstituted aminoacids were performed as previously reported (Schafmeister, C. E., J. Po,and G. L. Verdine, Journal of the American Chemical Society, 2000.122(24): p. 5891-5892; Walensky, L. D., et al., Science, 2004.305(5689): p. 1466-1470). Synthesis of (R) or (S)Fmoc-(2′-propenyl)alanine analogs were prepared using a new method asschematized in FIG. 6 and described below.

A solution of (R)-proline and KOH in isopropanol was prepared to whichbenzyl chloride was added and stirred at room temperature for 3 hr. Anacidic workup allowed for isolation of a precipitate in 89% yield. Thisproduct was dissolved in ice cold methylene chloride, to which thionylchloride and 2-aminobenzophenone was added to the reaction mixture andallowed to warm to room temperature with stirring over 10 hours. A basicworkup yielded (R)-2-[N—(N′-benzylprolyl)amino]benzophenone (BPB) in 81%yield. A solution of KOH in MeOH was poured into a stirred mixture ofBPB, Ni(NO₃)2-6H₂O, alanine in MeOH under inert gas at 40° C. Theresulting mixture was stirred for 2 h and an acidic workup yieldedAla-Ni(II)-BPB-complex in 91% yield. (Tetrahedron:Asymmetry 9 (1998)4249-4252)

The Ala-Ni(II)-BPB-complex was reacted with 3-bromo-1-propene in acetoneunder basic conditions to give a mixture of a Ni(II) complex of Schiffbase of (R)-BPB-(R)-trans-(2′-propenyl)alanine [(R)-2] and Ni(II)complex of Schiff base of (S)-BPB-(S)-trans-(T-propenyl)-alanine [(S)-2]with ratio 6:1.

After separation with silica gel column, diastereopure (R)-2 complexeswere obtained at 44% yield. The (R)-2 complexes were decomposed with 3NHCl/MeOH to afford (R)— (2′-propenyl)alanine as well as a chiral ligandwhich was extracted with DCM. After work up, (R)— (2′-propenyl)alaninewas protected with Fmoc-OSu to give the (R)-Fmoc-(T-propenyl)alaninewith 93% yield (two steps). (Tetrahedron 56 (2000) 2577-2582)

The modified polypeptide compounds were generated by replacing at leasttwo naturally occurring amino acids with the α,α-disubstitutednon-natural amino acids at discrete locations flanking either 2, 3 or 6amino acids, namely the “i, i+3,” “i, i+4” or “i, i+7” positions,respectively.

Locations for the non-natural amino acids and subsequent hydrocarbonstaple(s) were carefully chosen so as not to interfere with criticalGLP-1 receptor interactions or replace residues known to be critical forinsulinotropic activity (FIGS. 2 and 3).

The modified polypeptides, SAH-Ex and SAH-GLP1 (FIG. 7), were generatedusing solid phase Fmoc chemistry and ruthenium-catalyzed olefinmetathesis, followed by peptide deprotection and cleavage, purificationby reverse-phase high performance liquid chromatography, and chemicalcharacterization using LC/MS mass spectrometry and amino acid analysis.

Alternatively an established fragment-based approach can be pursued([Bray, B. L. Nature Reviews Drug Discovery, 2003. 2(7): p. 587-593;MYUNG-CHOL KANG, B. B., et al., Methods and compositions for peptidesynthesis, U.S.P.a.T. Office, Editor. Jan. 18, 2000 USA). In thisstrategy, the peptide is divided into 3 fragments, such that anN-terminal, central, and C-terminal portion are synthesizedindependently. These polypeptide fragments should be generated usingsolid phase Fmoc chemistry and ruthenium-catalyzed olefin metathesis onsuper-acid cleavable resins, which yields fully protected peptideshaving an Fmoc at the N-terminus, and either a C-terminal amide (for theC-terminal fragment) or a free carboxylate (for the central andN-terminal fragments). These fully protected fragments are purified byreverse-phase high performance liquid chromatography, followed bysequential deprotection, coupling, and purification, to yield the fulllength, fully protected polypeptides. Global deprotection, followed byreverse-phase high performance liquid chromatography will yield thefinal products, which can be characterized using LC/MS mass spectrometryand amino acid analysis.

Peptides were produced on an Apex 396 (Aapptec) automated peptidesynthesizer using Rink amide AM LL resin (EMD Biosciences, 0.2 mmol/gresin), at 50 mmol scale. The standard Fmoc protocol employed 2×10 mindeprotections in 20% piperidine/NMP followed by a pair of consecutivemethanol and dimethylformamide (DMF) washes. The incorporatednon-natural amino acids were treated with 4×10 min incubations in 20%piperidine/NMP to achieve complete deprotection. Amino acid coupling wasperformed using 0.4 M stock solutions of Fmoc-protected amino acids,0.67 M 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and 2 M N,N-diisopropyl ethylamine (DIEA),yielding 1 mL of 0.2 M active ester (4 equivalents). Coupling frequencyand incubation times were 2×30 min for standard residues, 2×45 min forthe olefinic non-natural amino acids, and 3×45 min for the residuefollowing a non-natural amino acid. The olefin metathesis step iscarried out by first swelling the resin with 1,2-dichloroethane followedby exposure to a 10 mM solution of bistricyclohexylphosphine)-benzylidene ruthenium(IV) dichloride (Grubbs'first generation catalyst) in 1,2-dichloroethane (0.20 mol % on thebasis of resin substitution) for 2 h. The stapling reaction is carriedout twice. The resin-bound peptide is then washed with1,2-dichloroethane three times and dried under a stream of nitrogen. Thecompleted peptide is cleaved from the resin and deprotected by exposureto trifluoroacetic acid (TFA)-based cleavage cocktails such asTFA/triisopropyl silane (TIS)/water (95%, 2.5%, 2.5%), and precipitatedwith methyl-tert-butyl ether followed by lyophilization. LyophilizedSAHB peptides are purified by reverse-phase HPLC by use of a C18 column.The compounds are characterized by LC/MS, with mass spectra obtained byelectrospray in positive ion mode.

Quantitation is achieved by amino acid analysis on a Beckman 6300high-performance amino acid analyzer.

Determining the Secondary Structure and Thermal Stability of theModified Polypeptides.

The alpha-helicity of stapled modified polypeptides was compared totheir unmodified counterparts by circular dichroism. CD spectra wereobtained on an Aviv spectropolarimeter at 20° C. using the followingstandard measurement parameters: wavelength, 190-260 nm; stepresolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1sec; bandwidth, 1 nm; path length, 0.1 cm. The alpha-helical content ofeach peptide was calculated by dividing the mean residue ellipticity[⊖]222_(obs) by the reported [⊖]222_(obs) for a model helical peptide(Forood, B., E. J. Feliciano, and K. P. Nambiar, PNAS, 1993. 90(3): p.838-842; J. Martin Scholtz, Biopolymers, 1991. 31(13): p. 1463-1470;Lawless, M. K., et al., Biochemistry, 1996. 35(42): p. 13697-13708) orusing, for example, the Aviv machine using CDNN software developed byBrohm in order to deduce five different secondary structure fractions(helix, parallel and antiparallel beta-sheet, beta-turn and randomcoil). Protein Engineering, 1992. 5(3); p. 191-195. Thermal stability isassessed by acquiring circular dichroism spectra across a broadtemperature range and determining the melting temperatures for eachconstrained peptide.

Optimization of the Biophysical and Biochemical Properties of theModified Polypeptides by Evaluating Diversified Modified PeptideLibraries Synthesized in High-Throughput Fashion.

High-throughput technologies were used to optimize the modifiedpolypeptides activities for cellular and in vivo studies. For example,an Apex 396 multichannel synthesizer (AAPPTEC; Louisville, Ky.) was usedto produce polypeptide libraries for biological evaluation. Thepolypeptide compounds were diversified by extension, truncation, oramino acid substitution across natural and select non-natural aminoacids, and differential staple localization were made to maximizedesirable biophysical and biochemical properties. The libraries weregenerated using high-throughput solid phase Fmoc chemistry andruthenium-catalyzed olefin metathesis and peptide deprotection andcleavage. Peptide purification was achieved by reverse phase C18 HPLC,and products characterized by LC/MS mass spectrometry and amino acidanalysis.

Protease Resistance Assays

In vitro proteolytic degradation of structured peptides was measured byLC/MS (Agilent 1200) using the following parameters: 20 μL injection,0.6 mL flow rate, 15 min run time consisting of a gradient of water(0.1% formic acid) to 20-80% acetonitrile (0.075% formic acid) over 10min, 4 min wash to revert to starting gradient conditions, and 0.5 minpost-time. The DAD signal was set to 280 nm with an 8 nm bandwidth andMSD set to scan mode with one channel at (M+2H)/2, +1 mass units and theother at (M+3H)/3, +1 mass units. Integration of each MSD signal yieldedareas under the curve of >10⁸ counts. Reaction samples were composed of5 μL peptide in DMSO (1 mM stock) and 195 μL of buffer consisting of 50mM phosphate buffer pH 7.4 containing 2 mM CaCl₂. Upon injection of the0 hr time point sample, 2 μL of 50 ng/μL chymotrypsin (Sigma) was addedand the amount of intact peptide quantitated by serial injection overtime. An internal control of acetylated tryptophan carboxamide at aconcentration of 100 μM was used to normalize each MSD data point. Aplot of MSD area versus time yielded an exponential decay curve andhalf-lives were determined by nonlinear regression analysis using Prismsoftware (GraphPad). The assay was also performed using trypsin andpepsin in their appropriate buffer conditions.

Plasma Stability and Pharmacokinetics

Peptides are incubated (5-10 μg) with mouse plasma at 37° C., injected(10, 20 mg/kg) by tail vein, or administered by oral gavage to maleC57/BL6 mice. Ex vivo and in vivo samples (withdrawn by retro-orbitalbleed) at various time intervals (e.g. 0.5, 1, 2, 4, 8, 12 hr; n=3 pertime point) are processed for quantitation by LC/MS analysis oravailable ELISA kits (e.g. Exendin-4 (Heloderma suspectum) EIA KitPhoenix Pharmaceuticals, Inc. EK-070-94). For ex vivo serum stabilitystudies, peptide half-lives are calculated by nonlinear regressionanalysis of exponential decay curves plotted using Prism software(Graphpad). In vivo plasma concentrations at the indicated time pointsare used to calculate plasma half-life, peak plasma levels, total plasmaclearance, and apparent volume of distribution using noncompartmentalanalysis. The derived protease-resistance and PK profiles serve as ameasure for selecting the most stable structured peptides for in vivoapplication.

GLP-1 Receptor Binding Assay.

Recombinant U2OS cells that stably express human GLP1 receptor fused tothe N-terminus of enhanced green fluorescent protein (EGFP) are employedin a GLP-1R Redistribution Assay (Thermo Scientific), performedaccording to the manufacturer's instructions. The GLP-1R assay is usedto screen for stapled insulinotropic polypeptide agonists that optimallytrigger internalization of GLP1R. GLP-1 is used as a reference compoundin the assay, and the ligands are assayed for their ability to induceGLP-1R internalization by a spot detecting image analysis algorithm. Thetranslocation of GLP-1R-EGFP is imaged on a fluorescence microscope. Thefilters are set for Hoechst (350/461 nm) and GFP/FITC (488/509 nm)(wavelength for excitation and emission maxima). The translocation isanalyzed on images taken with a 20× objective or higher magnification.The primary output in the GLP-1R Redistribution assay is the formationof spots in the cytoplasm. The data analysis therefore report an outputthat corresponds to number, area or intensity of spots in the cytoplasm.

GLP-1 Receptor Engagement Second Messenger Signaling Assays.

GLP-1R agonists have previously been shown to either prevent orameliorate experimental diabetes and preserve β-cell mass in multiplepreclinical models. Accordingly, the expression of markers important forthe response to ER stress in cells treated with vehicle alone or withstapled insulinotropic polypeptides can be examined. ER stress activatessignaling pathways invoked by the unfolded protein response involvingphosphorylation of many signaling protein such as ATF-4, XBP-1,phospho-eIF2a Ser51 (P[Ser51]-eIF2a), and CHOP (Druker, et. al., CellMetabolism, 2006 v4 p391-406). As such, Rat INS-1 or mouse MIN6insulinoma cells are treated with vehicle alone, thapsigargin, ortunicamycin in the absence or presence of serial dilutions of stapledinsulinotropic polypeptides for up to 4 hr. Alternatively, cultures areexposed to vehicle alone or to either H89, U0126, or LY294002 for 20 minprior to and during the 4 hr treatment with thapsigargin in the absenceor presence of stapled insulinotropic polypeptides. Total cell extractsare analyzed by immunoblotting for ATF-4, XBP-1, phospho-eIF2a Ser51(P[Ser51]-eIF2a), and CHOP with respect to time.

Glucose-Stimulated Insulin Release Cellular Assay.

INS-1 cells are cultured overnight in Dulbecco's modified Eagle's mediumcontaining 5 mM glucose and 10% fetal calf serum in the presence ofdiluent alone or various concentrations of stapled insulinotropicpolypeptides. After preincubation in the presence of 3.3 mM glucose,cells are then incubated in the presence of either 3 or 20 mM glucosefor 45 min at 37° C. The supernatant is then removed, centrifuged at 300g for 10 min, and assayed for insulin. To assess insulin content, cellsare extracted overnight in acid/ethanol mixture as described previously.The amount of insulin is then quantitated by ELISA (American LaboratoryProducts Company, Amin, et., al., The Journal of Pharmacology andExperimental Therapeutics, 2002, v303, p82-88).

In Vivo Monitoring of Serum Glucose and Insulin Levels in Response toInsulinotropic Peptide Treatment.

Db/db mice (C57BLKS/J-Leprdb/Leprdb) lacking the leptin receptor andtheir non-diabetic littermates are purchased at 4 weeks of age fromJackson Laboratories (Bar Harbor, Mass., USA). They are housed, two percage, and also fed ad libitum. The same mice are caged together for theduration of the study. After 2 days of acclimatization to ourfacilities, whole blood glucose concentrations, taken from aretro-orbital sinus, are determined using a Glucometer Elite (Bayer,Elkhart, Ind., USA). We administer insulinotropic polypeptides (e.g. 24nmol/kg exendin-4 i.p. daily) to 6 diabetic and 6 non-diabetic animalsthereafter (0700-0900 hours) and 10 diabetic and 10 non-diabetic animalsreceive NaCl i. p. This regimen is continued for 8 days. Animals areweighed daily and blood samples are taken from a retro-orbital sinus fordetermination of insulin and glucose concentrations. At the end of theregimen, fasting blood samples are obtained for these concentrations andwhole blood containing EDTA was assayed for HbA1c (Greig, et. al.,Diabetologia, 1999 v42, p45-50). The identical experiment is alsoperformed using oral gavage administration of insulinotropicpolypeptides (unmodified control and stapled derivatives).

Example 2 Singly and Doubly Stapled Exenatide Peptides DemonstrateEnhanced α-Helicity Compared to the Unmodified Template Peptide (FIG. 8)

To measure the effect of hydrocarbon stapling on the α-helical structureof exenatide peptides, we analyzed exenatide (Met14NorLeu), SAH-Ex(A),SAH-Ex (B), and SAH-Ex(A, B) by circular dichroism. The unmodifiedexenatide template peptide was predominantly unstructured in pH 7aqueous solution at 21° C., exhibiting less than 25% α-helicity. Allstapled derivatives displayed comparatively increased α-helical content.The insertion of either one or two hydrocarbon staples consistentlytransformed the circular dichroism spectra from a random coil patternwith a predominant single minimum at 204 nm to an α-helical contour withdouble minima at 208 and 222 nm.

Example 3 Singly and Doubly Stapled Exenatide Peptides DemonstrateEnhanced Thermal Stability Compared to the Unmodified Template Peptide(FIG. 8)

To assess the resistance of SAH-Ex peptides to thermal unfolding, weperformed circular dichroism studies across a 5-83° C. temperature rangefor all constructs. None of the peptides demonstrated classicalcooperative unfolding with increasing temperature, but instead showedincremental melting as a function of temperature. All SAH-Ex peptidesretained a greater degree of α-helicity across the entire temperaturerange. Whereas the slopes of exenatide (Met14NorLeu), SAH-Ex(A), andSAH-Ex(B) were similar, SAH-Ex(A, B) had a flatter sloped line,indicating that the α-helicity of the doubly stapled peptide helicitywas least effected by increasing temperature and reflecting a robustthermal stability.

Example 4 Singly and Doubly Stapled Exenatide Peptides DemonstrateEnhanced Proteolytic Stability at Neutral and Acidic pH Compared to theUnmodified Template Peptide (FIG. 9, 10)

A major limitation of peptides as therapeutics is their susceptibilityto rapid proteolytic degradation. Biologically active peptides such asexenatide and GLP-1 that are lengthy, partially or predominantlyunfolded, and replete with protease sites are particularly vulnerable.One of the potential benefits of a covalent crosslinking strategy toenforce peptide α-helicity is shielding of the vulnerable amide bondsfrom proteolysis. Because proteases require that peptides adopt anextended conformation to hydrolyze amide bonds, the structuralconstraint afforded by the hydrocarbon staple can render crosslinkedpeptides protease-resistant. To determine if hydrocarbon stapling couldprotect the 39-mer exenatide peptide from proteolysis, we subjectedexenatide (Met14NorLeu), and singly and doubly stapled derivatives todirect protease exposure in vitro. In the presence of 0.5 ng/μLchymotrypsin, Ex4 (25 μM) exhibited relatively rapid degradation, with ahalf-life of 38 minutes. In comparison, singly stapled SAH-Ex(A) andSAH-Ex(B) compounds longer half-lives of 94 and 128 minutes,respectively. The doubly stapled peptide SAH-Ex(A, B) displayed ahalf-life of 295 minutes, surpassing its singly stapled counterparts byup to 4-fold and the exenatide template peptide by 8-fold. Notably,double stapling itself had a stronger influence on proteolytic stabilitythan overall peptide α-helicity, as the doubly stapled peptide had alower percent α-helicity than the corresponding singly stapled peptidesyet exhibited superior protease resistance.

Peptides have poor oral bioavailability in part due to rapid acidhydrolysis in the proximal digestive tract. The enhanced proteaseresistance of stapled SAH-Ex peptides at neutral pH prompted us toexplore their stability under acidic conditions. Upon exposure to pepsinat 0.5 ng/μL, exenatide (Met14NorLeu exhibited rapid degradation, with ahalf-life of 13 minutes. Whereas N-terminal stapling did not enhancepepsin resistance in this example, stapling within the C-terminalportion of the peptide resulted in increasing the half-life to 81minutes, representing a more than 6-fold improvement over the unmodifiedtemplate peptide. As with chymotrypsin resistance at pH 7, thedouble-stapled peptide, SAH-Ex(A, B), exhibited striking pepsinresistance at pH 2, displaying a half-life of 172 minutes, representinga 13-fold improvement over the unmodified exenatide peptide template.

Example 5 A Doubly Stapled Exenatide Peptide Affords Similar SerumLevels Whether Administered by Intravenous Injection or Oral GavageDelivery (FIG. 11, 12)

The exendin-4 EIA kit (Phoenix Pharmaceuticals, Inc. EK-070-94)successfully recognized the template exenatide (Met14NorLeu) peptide andits doubly stapled derivative, enabling the measurement of SAH-Ex(A,B)levels in plasma (FIG. 11). In this competition ELISA, serially dilutedexenatide peptide derivatives compete with biotinylated controlexendin-4 peptide for an immobilized antibody; competition results areread-out by use of streptavidin-HRP-based detection of biotinylatedpeptide and effective competition is reflected by decreased absorbancedue to replacement of the biotinylated-exendin-4 by SAH-Ex peptide inthe immobilized antibody complex.

For in vivo detection studies, SAH-Ex(A,B) was dissolved in sterileaqueous 5% dextrose (1 mg/mL) and administered to 8-10 week old maleC57BL/6 mice (Jackson Laboratory, 3 animals per treatment group) byeither bolus tail vein injection (4 mg/kg, 125 mcg) or oral gavage (4mg/kg, 125 mcg). An additional cohort of mice was treated with vehiclealone. At 30 min after treatment, blood was withdrawn by retroorbitalpuncture in sufficient quantity to yield ˜200 μL of serum after clottingon ice and then centrifugation at 20,000 g for 1 min at 4° C. The serumsamples were purified using C18 solid-phase extraction columns (PhoenixPharmaceuticals) according to the manufacturer's protocol. Thewater-acetonitrile elution was lyophilized overnight and reconstitutedin 0.5 mL buffer from the ELISA kit. This reconstituted solution wassubsequently diluted serially by 10-fold and subjected to EIA analysisper the manufacturer's protocol. Strikingly, an equivalent amount ofSAH-Ex(A,B) was detected in the plasma of mice treated with eitherintravenous or oral gavage dosing, highlighting the remarkable capacityof double-stapling to transform an exenatide peptide into an orallybioavailable form that is absorbed by the gastrointestinal system intothe systemic circulation, a route of delivery previously unachievablefor unmodified exenatide (Gedulin et al., Int'l J Pharmaceuticals, 356(2008) 231-238). As a negative control, no signal was detected by EIA inserum from the vehicle-treated animals.

Example 6 Structural Determination of Stapled Insulinotropic Peptides

To define the explicit structure of stapled insulinotropic peptidesbound to the GLP-1 receptor x-ray crystallography methods will beapplied as described (Runge, S et al. 2008 J Biol Chem,283:11340-11347). Crystallization conditions for stapled insulinotropicpeptides are screened using 96-well sitting drop plates (Crystal Quick,Hampton Research) set up using a Phoenix crystallization robot. Initialconditions include HT Index Screen (Hampton Research), JSCG+ Suite(Qiagen) and Pro-Complex Suite (Qiagen). Screening around the best hit,including varying pH and salt and detergent concentrations, areperformed to identify the best condition for crystal growth. Oncegenerated, the crystals are removed, washed in the crystallizationbuffer, and subjected to mass spectroscopy to verify the presence ofpeptide within the crystal. The crystal is then soaked incyroprotectant, flash frozen, and stored in liquid nitrogen. Suitablecrystals are examined at the Argonne National Laboratory synchrotronfacility. Phases are obtained by molecular replacement followed by dataanalysis and refinement (Phaser, Phenix, and Coots software).

An alternative and complementary approach for structural analysisemploys ¹H-NMR analysis. Spectra of stapled insulinotropic peptides insolution are acquired on a Bruker Avance DRX spectrometer at 600 MHzequipped with a z-shielded gradient and triple resonance cryoprobe. Twodimensional DQF-COSY, TOCSY, and NOESY spectra are measured in 100% D₂Oand 90% H₂/10% D₂O. The TOCSY datasets are acquired with mixing times of40 and 80 ms and NOESY spectra with mixing times of 75, 100, 125 and 200ms. NMR data sets are processed with the NMRPipe spectral analysispackage and assignment of proton resonances is performed with Cara.Structure calculations are carried out with the program CYANA using thestandard protocol. The final structure family is comprised of the 20structures with the lowest target function and the best overall valuesfor chirality and stereochemistry measured with the programs WHATCHECKand PROCHECK_NMR. Structures are displayed and analyzed using theprograms PYMOL and MOLMOL.

The structural data are used to correlate stapled insulinotropic peptidestructure, individually and in complex with GLP-1R, with functionalactivity as evaluated in the assays described herein.

Example 7 Mechanism of Proteolytic Resistance Conferred by Insertion ofHydrocarbon Staples (FIG. 13)

The mechanism by which hydrocarbon stapling confers protease resistanceto a lengthy peptide therapeutic was explored using a gp41-derived HIV-1fusion inhibitor peptide template. Insertion of the two pairs ofolefinic non-natural amino acids without crosslinking (e.g. UnstapledAlpha Helix of gp41: UAH-gp41₍₆₂₆₋₆₆₂₎(A,B)) does not confer significantprotection from chymotrypsin proteolysis. However, upon olefinmetathesis, the corresponding doubly stapled analog,SAH-gp41₍₆₂₆₋₆₆₂₎(A,B), exhibited an 8-fold longer half-life thanUAH-gp41₍₆₂₆₋₆₆₂₎(A,B), indicating that the staples themselves arerequired to confer the striking protease resistance (FIG. 13A). UAH- andSAH-gp41₍₆₂₆₋₆₆₂₎(A,B) displayed similar circular dichroism meltingprofiles, with T_(m) values of 27° C. and 22° C., respectively.Temperature-dependent unfolding was reversible for both peptides, asevidenced by the overlapping repeat melting curves (FIG. 13B). Thesedata demonstrate that overall alpha-helical stabilization, which issimilar for the two constructs, does not account for the strikingprotease resistance of SAH-gp41₍₆₂₆₋₆₆₂₎(A,B). In addition, thereversibility of unfolding highlights the absence of peptideaggregation, which likewise cannot account for the striking proteaseresistance of SAH-gp41₍₆₂₆₋₆₆₂₎(A,B).

Comparative chymotrypsin degradation patterns of unmodified, singlystapled, doubly stapled, and 4-place substituted but unstapled peptidesrevealed that the N-terminal staple uniquely prevented proteolytichydrolysis of the cleavage site flanked by the staple, with nocorresponding M+18 species observed by LC/MS analysis (FIG. 13C). TheC-terminal staple slowed, rather than completely blocked, proteolysis atsites upstream of the staple. The 4-place substituted but unstapledderivative UAH-gp41₍₆₂₆₋₆₆₂₎(A,B) was not capable of blockingproteolysis at the position flanked by the N-terminal pair ofnon-natural amino acids, nor slow the rate of proteolysis as effectivelyas the C-terminal singly stapled peptide (T_(1/2) 77 min forSAH-gp41₍₆₂₆₋₆₆₂₎(B); T_(1/2) 36 min for UAH-gp41₍₆₂₆₋₆₆₂₎(A,B)). Thedoubly stapled peptide SAH-gp41₍₆₂₆₋₆₆₂₎(A,B) synergistically benefitedfrom the anti-proteolysis features of both the N-terminal and C-terminalstaples.

Comparative ¹H NMR analysis of SAH-gp41₍₆₂₆₋₆₆₂₎(A,B) and thecorresponding unmodified template peptide, T649v revealed that theindole protons (˜10.6 p.p.m) corresponding to the two N-terminaltryptophan residues of T649v are represented by two sharp peaks inT649v, consistent with fast exchange between multiple conformations(FIG. 13D). In contrast, the indole proton peaks in the ¹H NMR spectrumof SAH-gp41₍₆₂₆₋₆₆₂₎(A,B) are broadened and split, reflective of adiscretely structured N-terminus as a result of peptide stapling. Takentogether, these data indicate that the proteolytic advantage conferredby peptide double-stapling does not derive from mutagenesis of proteasecleavage sites, maximizing α-helicity alone, 4-place non-natural aminosubstitution, or peptide aggregation. Instead, we determined that thestriking protease resistance of doubly-stapled peptides is conferred bya combination of (1) decreased rate of proteolysis due to induction ofα-helical structure and (2) complete blockade of peptidase cleavage atsites localized within or immediately adjacent to the (i,i+4)-crosslinked segment.

Example 8 Structurally-Stabilized and Protease Resistant SAH-Ex andSAH-GLP Peptides Enhance Glucose-Stimulated Insulin Release (GSIS)

Stock solutions of SAH-Ex and SAH-GLP peptides were generated bydissolving the lyophilized powders in deionized water at 100 μM. Thefollowing treatment solutions were made for batch GSIS assays: 1.67 mMglucose, 16.7 mM glucose, 1.67 mM glucose+10 nM peptide, 16.7 mMglucose+10 nM peptide all in 1×KRB buffer, 2 mM CaCl₂, 0.05% BSA. Isletsisolated from wild-type mouse pancreas were washed 3 times with KRBbuffer and 5 islets per experimental tube were incubated in KRB buffercontaining 1.67 mM glucose for 30 min at 37° C. Islets were thenpelleted and the buffer replaced with the treatment solutions listedabove. Each experimental condition was examined in replicates of n=8.After 1 hour incubation at 37° C., islets were pelleted and thesupernatants collected for glucose stimulated insulin releasemeasurement. The pellets are solubilized to assess intracellular insulincontent to normalize for cell number. Insulin was measured by ELISAusing mouse insulin as a standard (Insulin ELISA Kit, cat. 80-INSMS-E01,ALPCO). For the insulin ELISA plots, there is a small increase in theamount of insulin released in response to escalation of the glucose dosefrom 1.67 mM to 16.7 mM. However, in response to the insulinsecretagogoues Ex4 and GLP1, and select SAH analogues, there is asubstantial increase in GSIS. These data highlight that hydrocarbonstapling, which improves the structural stability, protease resistance,and pharmacologic properties of Ex4 and GLP1, also preserves the keyfunctional GSIS activity of incretins.

ADDITIONAL REFERENCES

-   Walensky, L. D., Kung, A. L., Escher, I., Malia, T. J., Barbuto, S.,    et al. (2004) Activation of apoptosis in vivo by a    hydrocarbon-stapled BH3 helix. Science, 305(5689), 1466-1470.-   Bird, G. H., Bernal, F., Pitter, K., and Walensky, L. D. (2008)    Synthesis and biophysical characterization of stabilized    alpha-helices of BCL-2 domains. Methods Enzymol, 446, 369-386.-   Gavathiotis, E., Suzuki, M., Davis, M. L., Pitter, K., Bird, G. H.,    et al. (2008) BAX activation is initiated at a novel interaction    site. Nature, 455, 1076-1081.

INCORPORATION BY REFERENCE

All patents, patent applications, GenBank/PDB numbers, and publishedreferences cited herein are hereby incorporated by reference in theirentirety as if they were incorporated individually. While this inventionhas been particularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

1. A structurally-fortified insulinotropic polypeptide comprising analpha helix and one or more molecular tethers, wherein each moleculartether covalently couples a single pair of residues residing on thealpha helix of said polypeptide, thereby structurally fortifying theinsulinotropic polypeptide.
 2. The structurally-fortified insulinotropicpolypeptide of claim 1, wherein the insulinotropic polypeptide isexenatide, GIPP, GIP, GLP-1 precursor, GLP-1, GLP-2, GLP-1 (7-37),GLP-1-(7-36), liraglutide, taspoglutide, albiglutide or LY2189265. 3.The structurally-fortified insulinotropic polypeptide of claim 1,wherein the insulinotropic polypeptide is exenatide.
 4. Thestructurally-fortified insulinotropic polypeptide of claim 1, whereinthe number of molecular tethers is between 1-2, 2-3, 2-4, 2-5, 2-6, 2-7,2-8, 2-9 or 2-10.
 5. The structurally-fortified insulinotropicpolypeptide of claim 1, wherein at least one residue pair resides withinthe N-terminal half of the polypeptide.
 6. The structurally-fortifiedinsulinotropic polypeptide of claim 1, wherein at least one residue pairresides within the C-terminal half of the polypeptide.
 7. Thestructurally-fortified insulinotropic polypeptide of claim 1, comprisingat least one coupled residue pair within the C-terminal half of thepolypeptide and another coupled residue within the N-terminal half ofthe polypeptide.
 8. The structurally-fortified insulinotropicpolypeptide of claim 1, wherein the polypeptide corresponds to anexenatide having any one of SEQ ID NOs: 2 or 15-38.
 9. Thestructurally-fortified insulinotropic polypeptide of claim 1, whereinthe polypeptide corresponds to an GLP-1 having any one of SEQ ID NOs:39-62.
 10. The structurally-fortified insulinotropic polypeptide ofclaim 1, comprising a first molecular tether located at position (i,i+3), or (i, i+4) or (i, i+7) relative to the residue positions of thealpha helix of the polypeptide.
 11. The structurally-fortifiedinsulinotropic polypeptide of claim 1, comprising a first moleculartether located at position (i, i+3), or (i, i+4) or (i, i+7) relative tothe residue positions of the alpha helix of the polypeptide, and asecond molecular tether located at position (i, i+3), or (i, i+4) or (i,i+7) relative to the residue positions of the alpha helix of thepolypeptide, with the proviso that the first and second moleculartethers are not located at identical positions.
 12. Thestructurally-fortified insulinotropic polypeptide of claim 1, whereinthe fortified polypeptide possesses a half-life that is at least 2-foldgreater than the half-life of a non-fortified counterpart polypeptide.13-22. (canceled)
 23. A pharmaceutical composition comprising astructurally-fortified insulinotropic polypeptide of claim 1 and one ormore pharmaceutically acceptable excipients.
 24. The pharmaceuticalcomposition of claim 23, wherein the polypeptide template is exenatideof SEQ ID NO: 2 or a functional fragment or derivative thereof.
 25. Amethod for treating or preventing diabetes comprising administering atherapeutically effective amount of an insulinotropic polypeptide ofclaim
 1. 26. The method of claim 25, wherein the polypeptide isexenatide or a functional fragment or derivative thereof.
 27. A methodfor treating or preventing diabetes comprising administering atherapeutically effective amount of a pharmaceutical composition ofclaim
 23. 28. The method of claim 27, wherein the polypeptide isexenatide or a functional fragment or derivative thereof.
 29. A methodfor treating or preventing diabetes comprising administering atherapeutically effective amount of a structurally-fortifiedinsulinotropic polypeptide comprising an alpha helix and one or moremolecular tethers, wherein each molecular tether covalently couples asingle pair of non-natural amino acid residues residing on the alphahelix of said polypeptide.
 30. The method of claim 29, wherein theinsulinotropic polypeptide is exenatide, GIPP, GIP, GLP-1 precursor,GLP-1, GLP-2, GLP-1 (7-37), GLP-1-(7-36), liraglutide, taspoglutide,albiglutide or LY2189265, or a functional fragments or derivativesthereof.
 31. The method of claim 29, wherein the insulinotropicpolypeptide is exenatide or a functional fragment or derivative thereof.32-34. (canceled)
 35. The structurally-fortified insulinotropicpolypeptide of claim 1, comprising at least two molecular tethers whichare sequentially arranged and in a stitched configuration whereby theend of the first molecular tether and the beginning of the secondmolecular tether originate at a common residue in the polypeptide.